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      Location, Location, Location: Five Facts about Tissue Tropism and Pathogenesis

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      PLoS Pathogens
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          Abstract

          Infectious disease burden remains high [1]. An improved knowledge of host–microorganism interactions is key to delineating disease progression and developing new control and treatment modalities. Microorganisms may be pathogenic (able to cause disease) or non-pathogenic (unable to cause disease). Disease severity is influenced by host and pathogen factors; in particular, pathogens may express different virulence factors that modulate disease severity and progression. Within this context, the interaction of the pathogen with organ and tissue niches is especially important. Tropism refers to the ability of a given pathogen to infect a specific location. Organ or tissue tropism reflects the ability of a given pathogen to infect a specific organ or sets of organs. Some pathogens are broadly tropic, infecting all or most organs, while others are restricted to a given tissue or even to certain tissue niches. From this point of view, the ability of a pathogen to infect specific organs may vary over the course of the disease and could be active, pathogen-mediated, or passive, requiring, for example, a prior skin break or vector bite. Within tissue niches, intracellular pathogens may also preferentially infect specific organelles or intracellular sites. This article will focus on tissue tropism and its relationship to pathogenesis with examples from Staphylococcus aureus bacteria, Trypanosoma brucei protozoan parasites, and the influenza virus. Variations in Tropism S. aureus resides as a commensal in the nose and upper respiratory tract of 30% of individuals [2]. However, it also has the ability to cause a range of diseases, from localized skin abscesses to endocarditis, pneumonia, osteomyelitis, or disseminated infection [3]. Human African trypanosomiasis (HAT), caused by T. brucei rhodesiense and T. brucei gambiense, progresses through two stages of disease. In the first stage, the parasite remains in the peripheral blood and lymphatic system. The second stage is associated with the parasite crossing into the cerebrospinal fluid and ultimately into the brain parenchyma; however, the timing is still under discussion [4]. Parasites present in the cerebrospinal fluid and/or central nervous system (CNS) are also able to traffic back into the bloodstream [5]. Finally, seasonal influenza viruses mainly infect the upper respiratory tract, while pandemic influenza as well as some highly pathogenic avian influenza viruses (e.g., H5N1) have increased ability to infect the lower respiratory tract [6]. Influenza viruses can also infect extrapulmonary tissues, leading, for example, to conjunctivitis [7]. Impact of Tropism on Disease Severity As long as S. aureus resides as a commensal in the upper respiratory tract, it does not cause any symptoms [2]. In contrast, S. aureus bacteremia has a 15%–50% case fatality rate. Likewise, mortality from S. aureus infective endocarditis is 22%–66% [3]. Mortality in HAT is due to neurological symptoms that appear once the parasite enters the CNS, leading to approximately 30,000 deaths per year [8]. Slower progression to stage 2 HAT is associated with longer survival [9]. Seasonal influenza causes up to 500,000 deaths per year worldwide, while influenza pandemics can cause millions of deaths (up to 50 million deaths for the 1918 pandemic) [10]. The H5N1 avian influenza virus has about a 60% case fatality rate [6]. Impact of Tropism on Disease Transmission Pathogen location also strongly influences transmission to new hosts. Transmission of S. aureus from bacteremia appears to involve passage through the gastrointestinal tract followed by fecal spread [11]. In contrast, bacterial colonization on the skin and in the nose may facilitate person-to-person transmission [3]. In the case of T. brucei, CNS parasites are inaccessible to the tsetse fly vector. The presence of parasites in the bloodstream is essential for transmission [5]. The greater transmissibility of seasonal influenza viruses compared to avian influenza viruses may be due in part to the former’s superior ability to colonize the upper respiratory tract [10,12]. Particles in the upper respiratory tract are moved quickly towards the pharynx by the muco-ciliary escalator, whereas particles in the lower respiratory tract are cleared more slowly [13]. Mutations promoting soft palate infectivity also promoted transmission in a ferret influenza model. This environment may be more suitable to the generation of virus-containing droplets; tissue inflammation at this site may also stimulate sneezing, further enhancing transmissibility [14]. Mathematical modeling supports an association between lower infectivity rates deeper in the respiratory tract and enhanced transmission [13]. Impact of Tropism on Treatment Drug tissue penetration varies depending on chemical structure, formulation, and delivery. Treatment choices will therefore be influenced by the sites of infection. For example, S. aureus abscesses may be treated by incision and drainage alone, or with topical antibiotics. In contrast, bacteremia will require systemic antibiotics, and endocarditis may require surgery [3]. Likewise, infection of certain privileged sites complicates drug delivery. For example, stage 1 HAT is easier to treat than stage 2. Stage 2 drugs must be able to cross the blood–brain barrier and are associated with more severe side effects [4]. Elimination of circulating T. brucei parasites is insufficient to cure patients; infection relapses in the absence of clearance of CNS parasites [5]. Techniques to Study Mediators of Tropism Given the importance of tropism in the various scenarios discussed above, identifying the mediators of disease tropism has garnered considerable interest. Key mediators of disease tropism in staphylococcal infections include host characteristics such as immune status, concurrent infections, or medical procedures [3], while bacterial virulence factors include adhesins, metal acquisition genes, toxins, and immune evasion factors [15]. Blood–brain barrier crossing by T. brucei involves a combination of host and parasite factors. Many of the host factors promoting invasion are also involved in promoting T cell penetration into the brain parenchyma and in the pathogenesis of other infectious agents. These include TNFα, IFNγ, and CXCL10 [5]. Parasite factors are still poorly characterized but may involve proteases such as brucipain (T. brucei cathepsin L) [16]. Hemagglutinin receptor binding preference to alpha-2,3-linked versus alpha-2,6-linked sialosaccharides is the major determinant of upper versus lower respiratory tract influenza virus tropism, disease severity, and transmission [10,12]. Microenvironmental conditions surrounding the pathogen will alter virulence factor expression. It is, therefore, essential to study mediators of tropism in situ in models that will replicate disease conditions as much as possible. Human samples may be the best source where accessible, but humanized mouse models may represent a suitable compromise [17]. Intravital microscopy has provided significant insights into in vivo pathogen behavior but is limited in depth [4]. Non-invasive tracking methods using new luminescent markers help produce a dynamic time-resolved understanding of disease progression and lead to the identification of new or underestimated sites of infection [7]. Fluorescent markers can also be used to monitor the movement of an individual bacterium or parasite via photoconvertible markers [18], while functional microbial reporters can showcase in situ changes in conditions or pathogen physiology [19]. “Omics” methodologies are also expanding our understanding of disease tropism. Genomic, transcriptomic, proteomic, and metabolomic comparisons of different pathogen strains with variable tropism are regularly used to identify virulence factors (see for example [20], which showed a relationship between S. aureus toxicity and the ability to cause invasive disease) [9]. Dual host–pathogen RNA-seq using infected organs can also help describe host and pathogen responses in situ. New combinations of forward genetic screens and “omics” techniques are also being developed, such as high-throughput gene inactivation methodologies (RIT-seq) in T. brucei. These have been used to identify essential genes during in vitro culture and differentiation [8] but could readily be applied to identify parasite genes involved in host infection and tissue tropism. Finally, the increasing ease of genetic manipulation, including CRISPR/Cas9 technology, facilitates the necessary validation of the factors identified in these large-scale studies. Conclusions Disease prevention, monitoring, and treatment are mainstays of modern medicine. In this review, we highlighted the relationship between tissue tropism and disease severity, transmission, and treatment. Many infectious diseases, including sleeping sickness and staphylococcal infections, still lack effective vaccines. Vaccine development requires an understanding of the immune response required for protection, which is associated in part with the tissues targeted. Likewise, predicting disease progression from initial diagnosis remains a significant challenge. Identifying the site of penetration by infecting microorganisms and the tissues involved may help stratify patients and determine the appropriate course of treatment. This is especially important in the case of S. aureus infection to determine, for example, whether patients are at risk of disseminated infection. Overall, our understanding of the mediators of tissue tropism has progressed significantly; however, we cannot yet account for all the factors involved. Indeed, fine-scale differences in host tissue chemistry, metabolism, waste production, and local immune responses are still being identified. Moreover, while we usually know the ultimate tissue niche of a specific pathogen, we often do not understand the paths used to reach that location, as evidenced by the controversy surrounding the timing of brain penetration in T. brucei infection [4]. We are also beginning to appreciate that tropism is dynamic rather than static. Large-scale application of new technologies should facilitate continuing advances in this field and, ultimately, lead to the discovery of new methods to target these pathogens.

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          High-throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome.

          African trypanosomes are major pathogens of humans and livestock and represent a model for studies of unusual protozoal biology. We describe a high-throughput phenotyping approach termed RNA interference (RNAi) target sequencing, or RIT-seq that, using Illumina sequencing, maps fitness-costs associated with RNAi. We scored the abundance of >90,000 integrated RNAi targets recovered from trypanosome libraries before and after induction of RNAi. Data are presented for 7435 protein coding sequences, >99% of a non-redundant set in the Trypanosoma brucei genome. Analysis of bloodstream and insect life-cycle stages and differentiated libraries revealed genome-scale knockdown profiles of growth and development, linking thousands of previously uncharacterized and "hypothetical" genes to essential functions. Genes underlying prominent features of trypanosome biology are highlighted, including the constitutive emphasis on post-transcriptional gene expression control, the importance of flagellar motility and glycolysis in the bloodstream, and of carboxylic acid metabolism and phosphorylation during differentiation from the bloodstream to the insect stage. The current data set also provides much needed genetic validation to identify new drug targets. RIT-seq represents a versatile new tool for genome-scale functional analyses and for the exploitation of genome sequence data.
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            The soft palate is an important site of adaptation for transmissible influenza viruses

            Influenza A viruses (IAV) pose a major public health threat by causing seasonal epidemics and sporadic pandemics. Their epidemiological success relies on airborne transmission (AT) from person-to-person; however, the viral properties governing AT of IAV are complex. IAV infection is mediated via binding of the viral hemagglutinin (HA) to terminally attached α2,3 or α2,6 sialic acids (SA) on cell surface glycoproteins. Human IAV preferentially bind α2,6-linked SA while avian IAV bind α2,3-linked SA on complex glycans on airway epithelial cells 1,2 . Historically, IAV with preferential association with α2,3-linked SA have not transmitted efficiently by the airborne route in ferrets 3,4 . In this study, we observed efficient AT transmission of a 2009 pandemic H1N1 virus (H1N1pdm) engineered to preferentially bind α2,3SA. AT was associated with rapid selection of virus with a change at a single HA site which conferred binding to long-chain α2,6SA, without loss of α2,3SA binding. The transmissible virus emerged in experimentally infected ferrets within 24 hours post-infection and was remarkably enriched in the soft palate (SP), where long-chain α2,6SA predominate on the nasopharyngeal surface. Importantly, presence of long-chain α2,6SA is conserved in ferret, pig and human SP. Using a “loss-of-function” approach with this one virus, we demonstrate that the ferret SP, a tissue not normally sampled, rapidly selects for transmissible IAV with human receptor (α2,6SA) preference. Receptor-binding specificity is an important determinant of host-range restriction and transmission of IAV 4,5 and reviewed in 6 . The ability of zoonotic IAV for AT increases their pandemic potential 7 . Recently, several investigators have attempted to identify viral determinants of AT by generating transmissible H5 and H7 avian IAV 8-10 . We approached the question differently and used an epidemiologically successful IAV in which we altered receptor preference from the human (α2,6SA) to the avian receptor (α2,3SA). We previously generated H1N1pdm virus variants, with highly specific binding to either α2,6 or α2,3 SA, referred to as α2,6 or α2,3 H1N1pdm respectively 11 . The α2,3 H1N1pdm virus was generated by introducing four amino acid (aa) mutations in the receptor binding site (RBS) of HA (D187E, I216A, D222G, and E224A) 11 . Unexpectedly, the α2,6 and α2,3 H1N1pdm viruses transmitted via AT equally well in ferrets (Fig.1, Supplemental Table1) and with a similar efficiency as observed previously for wild-type H1N1pdm virus 12-15 . A delay in peak viral shedding was noted in the airborne-contact (AC) animals in the α2,3 virus group (red arrows, Fig.1) suggesting that the virus evolves prior to transmission. Deep sequence analysis of viral RNA (vRNA) extracted from nasal washes (NW) of α2,3 H1N1pdm virus-infected ferrets revealed a mixed population at aa 222 (H1 numbering) with the engineered glycine (G) and wild-type aspartic acid (D), while the other three engineered changes in the HA were retained (Fig.2a, Supplemental Table2). Interestingly, the vRNA from the NW of AC ferrets contained only the G222D HA mutation (Fig.2a, Supplemental Table2), suggesting that this sequence at aa 222 in the α2,3 H1N1pdm virus was associated with AT. The virus inoculum did not contain a mixture at this residue (Fig.2a) and associated changes were not observed in the neuraminidase gene (Supplemental Table3). A D222G change in the 2009 H1N1pdm virus HA has occurred in natural isolates and reports suggest an association with increased virulence in humans and no effect on AT 16-18 . Theoretical structural analysis suggest that the G222D reversion makes the RBS better suited to bind α2,6SA while retaining contacts with α2,3SA via glutamic acid at aa 187 (Extended Data Fig.1). Glycan binding data corroborated this structural prediction because the G222D mutation caused no change in α2,3SA binding but substantially increased binding to long-chain α2,6SA (Fig.2b). Previous reports have demonstrated the importance of α2,6SA binding for transmission 4,5,19 . We now demonstrate conclusively that AT requires gain of long-chain α2,6SA binding and, contrary to previous suggestions 4 , loss of α2,3SA binding is not necessary. The presence of a distinct and identifiable HA sequence in the transmissible virus allowed us to determine whether it emerges in a specific area of the respiratory tract of experimentally infected ferrets. Tissue sections and samples from the upper and lower respiratory tract were collected on several days post-infection (DPI) from groups of 3 ferrets infected with the α2,3 H1N1pdm virus. Virus was detected in all ferrets and all samples (Extended Data Fig.2). Deep sequencing of vRNA from both the upper and lower respiratory tract revealed a mixed population at residue 222 (Fig.3). Surprisingly, vRNA from the SP was remarkably and uniquely enriched for the G222D virus on 1 DPI and ≥90% of the sequences encoded 222D at 3 DPI (Fig.3c). All other engineered mutations were maintained (Extended Data Fig.3). These data suggest that the G222D revertant virus was actively selected in the ferret SP. To determine whether the rapid enrichment of G222D revertant virus in the SP was responsible for infection of the AC animal, we performed an AT study where naïve ferrets were exposed to experimentally infected donor ferrets for only 2 days. Surprisingly, even within this shortened exposure time, two AC animals shed virus and 3 out of 4 AC animals seroconverted (Extended Data Fig.4 and Supplemental Table1). Sequence analysis of vRNA from the two AC animals with detectable virus in the NW revealed presence of the G222D revertant. These data suggest that the selection of the α2,3 H1N1pdm virus with the 222D sequence occurs within 3 DPI in the donor ferret and that the AC ferrets were possibly infected with virus originating in the SP because there was nearly complete selection of the G222D mutant by 3 DPI in this tissue. The SP, with mucosal surfaces facing the oral cavity and nasopharynx, is not usually examined in animal models of influenza. To understand what drives the enrichment of the long-chain α2,6SA-binding α2,3 H1N1pdm virus at this site, we stained the SP with lectins specific for α2,6 or α2,3 SA (Extended Data Fig.5). The ciliated respiratory epithelium (RE) and mucus secreting goblet cells in the RE and submucosal glands (SMG) contained α2,6SA (SNA staining) (Extended Data Fig.5). Expression of α2,3SA (MAL II staining) was present in the connective tissue underlying the RE and in the serous cells of the SMG. Using a purified HA protein (SC18) that selectively binds long-chain α2,6SA 20 , we found high expression of long-chain α2,6SA in the SP compared to the trachea and lungs of ferrets (Fig.4, Extended Data Fig.6). A recent report detailing the glycan profile of the ferret respiratory tract confirms that the SP abundantly expresses α2,6 sialylated LacNAc structures 21 , similar to the long-chain α2,6SA recognized by SC18 HA. Interestingly, both the RE and olfactory epithelium (OLF) from the nasal turbinates (NT) of ferrets expressed high levels of long-chain α2,6SA, but the RE of the NT was not enriched for G222D mutant (Fig 3b, Extended Data Fig.6). These data suggest that the SP is unusual in driving selection for the G222D virus. To determine the relevance for humans, we evaluated the expression of long-chain α2,6SA in the SP of humans and pigs. Interestingly, expression of long-chain α2,6SA was conserved on the RE and goblet cells of the SP of both species (Fig.4). In addition, staining with plant lectins specific for α2,6 or α2,3SA (Extended Data Fig.7) revealed that α2,6SA were present on the nasopharyngeal surface and SMG of both pigs and humans. Expression of α2,3SA was detected in the basal cells of the oral surface and on the nasopharyngeal surface of the human SP; these findings are consistent with reports describing the SA distribution in the human nasopharynx 22 . Other investigators have also reported replication of seasonal and pandemic IAV in tissue sections obtained from the human nasopharynx 23 . Taken together these data highlight the importance of the nasopharynx, of which the SP forms the floor, as a site for host adaptation of IAV. IAV infection of the SP may contribute to AT by providing a mucin-rich microenvironment for generation of airborne virus during coughing, sneezing or breathing. Infection with α2,3 H1N1pdm virus resulted in severe inflammation and necrosis of the RE cells and SMG in the SP (Extended Data Fig.8). Since the SP is innervated by the trigeminal nerve, inflammation of this tissue could stimulate sneezing. Alternatively, the SP may be the site where infection is initiated during AT; therefore binding to this tissue would provide a fitness advantage. These results, albeit with one virus enhance our understanding of the properties necessary for AT of IAV in the ferret model. Loss of α2,3SA specificity is not necessary but gain of long-chain α2,6SA binding is critical for efficient AT of IAV. H7N9 viruses from China show dual receptor binding but variable AT efficiency in ferrets 24,25 . Interestingly, the 1918 H1N1 virus (A/New York/1/18), which has a similar SA binding preference as the α2,3 H1N1pdm virus, did not transmit via the airborne route or adapt within the ferret host 4 , suggesting that H1N1pdm virus may be unusual for this rapid adaptation. However, Pappas et al recently reported the detection of a mutation that enhanced α2,6SA binding in nasal washes of ferrets infected with avian H2 viruses 26 , demonstrating that rapid adaptation of IAV to gain human receptor preference occurs in other IAV subtypes as well. Studies with transmissible H5 viruses suggest that increased pH and thermal stability of the HA enhance AT 8,9,27 . Although we did not observe adaptive mutations in the HA stalk of the α2,3 H1N1pdm virus, perhaps because H1N1pdm HA is already adapted to humans, a mixed population was observed at four lysine residues around the RBS (Extended Data Fig.9, Supplemental Table2). Some are known to be egg adaptive mutations 28 or are components of the proposed positively charged ‘lysine fence’ around the base of the RBS, positioned to anchor the N-acetylneuraminic acid and galactose sugar of α2,3 and α2,6SA glycans 29 . Interestingly, the lysine residues were restored in the vRNA isolated from NW of AC ferrets and the SP of experimentally infected ferrets (Extended Data Fig.9,10). Taken together with our previously published data, long-chain α2,6SA binding and a highly active neuraminidase contribute to the AT of the H1N1pdm virus 12,30 . Importantly, we have identified the previously overlooked SP as an important site of isolation of transmissible virus and perhaps the initial site of infection. Analysis of the replicative fitness of IAV in this tissue may be warranted in assessment of their pandemic potential. Materials and Methods Ethics Statement and Animal Studies This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The National Institutes of Health Animal Care and Use Committee (ACUC) approved the animal experiments that were conducted. All studies were conducted under ABSL2 conditions and all efforts were made to minimize suffering. In our animal study protocol, we state that the number of animals in each experimental group varies, and is based on our prior experience. We use the minimum number of animals per group that will provide meaningful results. Randomization was not used to allocate animals to experimental groups and the animal studies were not blinded. Virus Rescue The 2009 H1N1pdm virus used in this study is A/California/07/2009. Generation and characterization of the α2,3 H1N1pdm and α2,6 H1N1pdm viruses has been described previously 11 . Genomic sequencing and dose dependent glycan binding assays confirmed the identity and receptor specificity of viruses generated by reverse genetics. All experiments were performed using viruses passaged no more than 3 times in MDCK cells or eggs. Ferret Transmission Study All ferrets (Mustela putorius furo) were screened by hemagglutination inhibition (HAI) assay prior to infection to ensure that they were naïve to seasonal influenza A and B viruses and the viruses used in this study. The transmission studies were conducted in adult ferrets as previously described 12 , male and female ferrets were used in a 3:1 ratio and sample size was based on the capacity of the transmission cages. Ferrets reaching 15-20% weight loss were provided with enriched diet and monitored closely by veterinarian staff for altered behavior. Environmental conditions inside the laboratory were monitored daily and were consistently 19±1°C and 56±2% relative humidity. The transmission experiments were conducted in the same room, to minimize any effects of caging and airflow differences on aerobiology. On day 0 four animals were infected intranasally with 106 TCID50 of either α2,3 H1N1pdm or α2,6 H1N1pdm virus and placed into the transmission cage. Twenty-four hours post-infection, a naïve animal (airborne-contact or AC) was placed into the transmission cage on the other side of a perforated stainless steel barrier. The AC ferrets were always handled before the infected ferrets. Nasal washes were collected and clinical signs were recorded on alternate days from days 0 to 14. Great care was taken during nasal wash collections and husbandry to ensure that direct contact did not occur between the ferrets. On 14 days post-infection (DPI), blood was collected from each animal for serology. The shortened exposure time study was done similarly except 48 hours after the naïve recipient animal (Airborne contact - AC) was placed into the transmission cage the ferrets were separated into micro-isolator cages. Infected ferrets were sacrificed on 7 DPI and the AC animals were sacrificed on 21 DPI. The AC animals were always handled before infected ferrets and all husbandry tools were decontaminated three times between handling of each AC animal. Dose dependent direct binding of influenza viruses To determine the receptor specificity of the G222D α2,3 H1N1pdm virus, virus from the nasal wash of a single AC animal on day 6 post-exposure was propagated once in MDCK cells. This virus stock was inactivated with betapropiolactone (BPL) and the hemagglutination titer was determined. For the glycan binding assay, 50μl of 2.4 μM biotinylated glycans were added to wells of streptavidin-coated high binding capacity 384-well plates (Pierce) and incubated overnight at 4°C. The glycans included were 3′SLN, 3′SLN-LN, 3′SLN-LN-LN, 6′SLN and 6′SLN-LN (LN corresponds to lactosamine (Galβ1-4GlcNAc) and 3′SLN and 6′SLN respectively correspond to Neu5Acα2-3 and Neu5Acα2-6 linked to LN) that were obtained from the Consortium of Functional Glycomics (www.functionalglycomics.org). The inactivated G222D virus was diluted to 250 μl with 1X PBS + 1% BSA. 50 μl of diluted virus was added to each of the glycan-coated wells and incubated overnight at 4 °C. This was followed by three washes with 1X PBST (1X PBS + 0.1% Tween-20) and three washes with 1X PBS. The wells were blocked with 1X PBS + 1% BSA for 2 h at 4 °C followed by incubation with primary antibody (ferret anti – CA07/09 antisera; 1:200 diluted in 1X PBS + 1% BSA) for 5 h at 4 °C. This was followed by three washes with 1X PBST and three washes with 1X PBS. Finally, the wells were incubated with the secondary antibody (goat anti-ferret HRP conjugated antibody from Rockland; 1:200 diluted in 1X PBS + 1% BSA). The wells were washed with 1X PBST and 1X PBS as before. The binding signals were determined based on the HRP activity using the Amplex Red Peroxidase Assay (Invitrogen) according to the manufacturer's instructions. Negative controls were uncoated wells (without any glycans) to which just the virus, the antisera and the antibody were added and glycan coated wells to which only the antisera and the antibody were added. Ferret Replication We evaluated the replication kinetics of the α2,3 H1N1pdm virus in the respiratory tract of 6-8 month old male ferrets as previously described 11 . Briefly, all ferrets were screened prior to infection by HAI assay to ensure that they were naïve to seasonal influenza A and B viruses. Animals were infected intranasally with 106 TCID50 of α2,3 H1N1pdm virus in 500μl. Tissues were harvested to assess viral titers. Tissues were weighed and homogenized in Leibovitz's L-15 (L-15, Invitrogen) at 5% (nasal turbinates and trachea) or 10% (lung) weight per volume (W/V). The soft palate was homogenized in 1 mL of L-15. Clarified supernatant was aliquoted and titered on MDCK cells. The 50% tissue culture infectious dose (TCID50) per gram of tissue was calculated by the Reed and Muench method 31 . Influenza A virus Full genome Sequencing The influenza A genomic RNA segments were simultaneously amplified from 3 μl of purified RNA (from homogenized ferret tissue) using a multi-segment RT-PCR strategy (M-RTPCR) 32 . In a separate reaction, each HA segment was amplified using HA-specific primers (swH1ps-1A-F: 5′-AGCAAAAGCAGGGGAAAACAAAAGCAAC-3′;swH1ps-1777A-R: 5′-AGTAGAAACAAGGGTGTTTTTCTCATGC-3′). Analysis of influenza viral RNA from ferret trachea and region of nasal turbinates enriched for respiratory epithelium (RE), between the canine and 2nd premolar teeth, was collected from tissue stored in RNAlater (Ambion) and total RNA was extracted using RNAeasy Kit (Qiagen). For these samples, nested HA-specific small amplicons were generated using HA-specific PCR primers (Outer primer pair H1-399F: 5′-AGCTCAGTGTCATCATTTGAAAG-3′ and H1-961R: 5′-TGAAATGGGAGGCTGGTGTT-3′; and inner primer pair H1-468 F:5′-AACAAAGGTGTAACGGCAGC-3′ and H1-884R: 5′-AATGATAATACCAGATCCAGCAT-3′). Illumina libraries were prepared from M-RTPCR products and from HA-specific RT-PCR products using the Nextera DNA Sample Preparation Kit (Illumina, Inc., San Diego, CA, USA) with half-reaction volumes. After PCR amplification, 10 μl of each library derived from M-RTPCR products was pooled into a 1.5 mL tube; separately, 10 μl of each library derived from HA-specific amplicons was pooled into a 1.5 mL tube. Each pool was cleaned two times with Ampure XP Reagent (Beckman Coulter, Inc., Brea, CA, USA) to remove all leftover primers and small DNA fragments. The first and second cleanings used 1.2× and 0.6× volumes of Ampure XP Reagent, respectively. The cleaned pool derived from M-RTPCR products was sequenced on the Illumina HiSeq 2000 instrument (Illumina, Inc.) with 100-bp paired-end reads, while the cleaned pool derived from HA-specific amplicons was sequenced on the Illumina MiSeq v2 instrument with 300-bp paired-end reads. For additional sequencing coverage, and the HA specific small amplicons, samples were re-sequenced using the Ion Torrent platform. M-RTPCR products were sheared for 7 min, and Ion-Torrent-compatible barcoded adapters were ligated to the sheared DNA using the Ion Xpress Plus Fragment Library Kit (Thermo Fisher Scientific, Waltham, MA, USA) to create 400-bp libraries. Libraries were pooled in equal volumes and cleaned with the Ampure XP Reagent. Quantitative PCR was performed on the pooled, barcoded libraries to assess the quality of the pool and to determine the template dilution factor for emulsion PCR. The pool was diluted appropriately and amplified on Ion Sphere Particles (ISPs) during emulsion PCR on the Ion One Touch 2 instrument (Thermo Fisher Scientific). The emulsion was broken, and the pool was cleaned and enriched for template-positive ISPs on the Ion One Touch ES instrument (Thermo Fisher Scientific). Sequencing was performed on the Ion Torrent PGM using a 318v2 chip (Thermo Fisher Scientific). Deep sequencing analysis Deep sequencing preparation, collection, and analysis was conducted by investigators who were blinded to the experimental groups. For virus sequence assembly, all sequence reads were sorted by barcode, trimmed, and de novo assembled using CLC Bio's clc_novo_assemble program (Qiagen, Hilden, Germany). The resulting contigs were searched against custom full-length influenza segment nucleotide databases to find the closest reference sequence for each segment. All sequence reads were then mapped to the selected reference influenza A virus segments using CLC Bio's clc_ref_assemble_long program. Minor allele variants were identified using FindStatisticallySignificantVariants (FSSV) software (http://sourceforge.net/projects/elvira/). The FSSV software applies statistical tests to minimize false-positive SNP calls generated by Illumina sequence-specific errors (SSEs) described in 33 . SSEs usually result in false SNP calls if sequences are read in one sequencing direction. The FSSV analysis tool requires observing the same SNP at a statistically significant level in both sequencing directions. Once a minimum minor allele frequency threshold and significance level are established, the number of minor allele observations and major allele observations in each direction and the minimum minor allele frequency threshold are used to calculate p-values based on the binomial distribution cumulative probability. If the p-values calculated in both sequencing directions are less than the Bonferroni-corrected significance level, then the SNP calls are accepted. A significance level of 0.05 (Bonferroni-corrected for tests in each direction to 0.025) and a minimum minor allele frequency threshold of 3% were applied for this analysis. Differences in the consensus sequence compared to the reference sequence were identified using CLC Bio's find_variations software. The identified consensus and minor allele variations were analyzed by assessing the functional impact on coding sequences or other regions based on overlap with identified features of the genome. For each sample, the reference sequence was annotated using VIGOR software 34 , and then the variant data and genome annotation were combined using VariantClassifier software 35 to produce records describing the impacts of the identified variations. Lectin and Immuno Histochemistry Lectin histochemistry was performed as described previously for plant lectins 36 and purified HA protein 37 . For plant lectin staining, the soft palate was subjected to microwave-based antigen retrieval using a citrate buffer and was then incubated with FITC-conjugated Sambucus nigra agglutinin (SNA) and biotinylated Maackia amurensis agglutinins (MAL II) lectins (Vector Laboratories), followed by a streptavidin-Alexa-Fluor594 conjugate (Invitrogen). For SC18 staining, the tissue sections were incubated with precomplexed purified His-tagged SC18 HA protein, mouse anti-His antibody (Abcam), and goat anti-mouse IgG secondary antibody conjugated to Alexa-Fluor 488 (Molecular Probes) at a 4:2:1 ratio. Nuclei were counter stained with DAPI (Vector Laboratories) and sections were mounted with either ProLong Gold anti-fade reagent (Invitrogen) or Fluoromount-G (Southern Biotech). Images were captured either on an Olympus BX51 microscope with an Olympus DP80 camera or a Leica SP5 confocal microscope. Ferret nasal turbinate biopsy samples were obtained from an uninfected ferret 8 months old as follows: the head was dissected sagittally to expose two halves of the ferret nasal turbinates, biopsy of turbinates between the canine and 2nd premolar represented respiratory epithelium (RE) and biopsy of turbinates at the molar tooth represented olfactory epithelium (OLF). A schematic depicting these two areas is shown in extended data figure 5H. Pig soft palate tissue sections were a kind gift from Dr. XJ Meng (Virginia Tech College of Veterinary Medicine) and Dr. Pablo Pineyro (Iowa State University). Pig soft palate tissues were collected from four 56 day-old mixed-breed commercial swine and fixed in 10% formalin. Soft palate tissues from four adult cadavers were obtained from the Maryland State Anatomy Board, Department of Heath and Mental Hygiene in Baltimore, MD. Extended Data Extended Data Fig. 1 Amino acids in the receptor binding site of H1N1pdm HA that bind to α2,3 and α2,6 glycans Ribbon diagrams of the 2009 H1N1pdm HA receptor binding pocket interacting with an α2,6 sialic acid in the pocket (a), an α2,3 H1N1pdm HA with α2,3 glycan (b), or α2,3 G222D revertant H1N1pdm HA and α2,6 sialic acid (c). Extended Data Fig. 2 Replication of α2,3 H1N1pdm virus in ferret respiratory tract We confirmed that the α2,3 H1N1pdm virus replicated to high titers on days 1, 3, and 5 in different parts of the ferret respiratory tract. Each tissue homogenate is highlighted with a dashed-circle, the gray circles represent washes. Each point represents a single animal. The horizontal black line indicates the mean viral titer on a given day. Extended Data Fig. 3 Stability of engineered mutations in viruses replicating in the soft palate Deep sequencing of the HA gene segment from virus populations in the soft palate from 1, 3, 5, and 7 DPI reveals a rapid change at position 222, but no change in the other engineered sites. The engineered sites are highlighted in blue, while the wild-type nucleotide is in orange. Each bar represents a single animal. Extended Data Fig. 4 Airborne transmission of α2,3 H1N1pdm virus after 48 hour exposure time One ferret in each pair was infected with 106 TCID50 of the indicated virus, a naïve ferret (referred to as airborne-contact or AC) was introduced into the adjacent compartment 24 hours later. The AC animal was removed from the transmission cage on day 3 post-infection as indicated by the black arrow. Nasal secretions were collected every other day for 14 days. Viral titers from the nasal secretions are graphed for each infected or AC animal. The gray shading indicates the exposure time between the infected and AC animals. Extended Data Fig. 5 Influenza receptor distribution on ferret soft palate Hematoxylin and eosin (H&E) staining of the soft palate from an uninfected ferret highlights the nasopharyngeal and oral surfaces. Scale bar is 1.25mm. (a) Areas highlighted in parts b-g are marked with dashed line shapes: square – nasopharyngeal surface (b and e), circle - submucosal gland (d and g) and triangle - oral surface (c and f). H&E staining of these regions, reproduced from Figure 4A-C in the main text, are shown in b-d. Staining with plant lectins specific for α2,6 SA (SNA) and α2,3 SA (MAL II) are shown in e-g. Scale bars are 100μm in images b-g. Extended Data Fig. 6 SC18 staining of ferret respiratory tissues Sections of ferret trachea (a) lung (b), soft palate (c and d), biopsy of nasal turbinate tissue with respiratory epithelium (RE) (f) and olfactory epithelium (OLF) (g) were stained with purified SC18 HA protein to identify areas expressing long-chain α2,6 SA. Illustration of ferret head (sectioned along the midline) highlighting the anatomical locations of RE and OLF tissues is depicted in (h). Goblet cells on the respiratory epithelium of the soft palate (nasopharyngeal surface) also stained positive for SC18 (d). Absence of SC18 staining after sialidase A treatment (e) indicates the high specificity of SC18 for the respiratory epithelium of the soft palate. All scale bars are 100μm unless indicated. Extended Data Fig. 7 Influenza receptor distribution on pig and human soft palate Pig (a-c) and human (g-i) soft palate tissues were stained with plant lectins SNA and MALII which are commonly used as markers for α2,6 and α2,3 sialic acid respectively. Sialidase A treated control was run for each sample to ensure specificity of plant lectins and are displayed in panels (d-f and j-l). Expression of α2,6 sialic acids (SNA staining) is found on the ciliated respiratory epithelium and goblet cells of the nasopharyngeal surface and in the submucoasl glands of both the pig and human soft palate. Expression of α2,3 sialic acids is low in the pig soft palate and found primarily in goblet cells and submucosal glands. In the human soft palate, MALII (α2,3 sialic acids) staining sensitive to sialidase A treatment is found in the goblet cells and respiratory epithelium of the nasopharyngeal surface and in the basal cells of the oral surface. MALII staining in the submucosal glands was not sensitive to sialidase A treatment. Scale bars are 100μm in all images. Extended Data Fig. 8 Pathology of the soft palate during infection with α2,3 H1N1pdm The soft palate was removed from 3 ferrets infected with α2,3 pH1N1 virus on 7 DPI. The tissue sections were stained with hematoxylin and eosin. Black arrows indicate the ciliated respiratory epithelium of the soft palate tissue (nasopharyngeal surface). Scale bars are 100μm in all images. Extended Data Fig. 9 Quasispecies in putative lysine fence Deep sequencing analysis of the α2,3 H1N1pdm inoculum revealed a mixed population at 4 lysine residues surrounding the receptor binding site of the HA protein. The lysine fence was restored in viruses from the nasal wash of AC animals from 6 days post-exposure (DPE). Each bar represents a single animal, and each amino acid (aa) that contained a quasispecies is indicated. Extended Data Fig. 10 Quasispecies of lysine fence in various ferret respiratory tissue sections Deep sequencing of viruses from respiratory tissues of ferrets infected with α2,3 H1N1pdm. Viruses populations from the soft palate (a), nasal wash (b), nasal turbinates (c), trachea (d), bronchoalveolar lavage (BAL) (e), or lung sections (f) were analyzed and the proportion of lysine, glutamic acid, or asparagine are presented. Each bar represents a single animal. The lung section is an average of the right middle lung lobe and a portion of the left caudal lung tissue. Supplementary Material 1
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              Differential Expression and Roles of Staphylococcus aureus Virulence Determinants during Colonization and Disease

              INTRODUCTION Staphylococcus aureus is a Gram-positive bacterium that causes both community- and hospital-acquired infections (1). Approximately 20 to 30% of the population is reported to be permanent carriers in the nose and 60% are intermittent carriers (2). Nasally colonized individuals are at increased risk for developing an infection with their colonizing isolate (2, 3). S. aureus infections range from mild skin and soft tissue infections to more severe infections, including bacteremia, sepsis, and osteomyelitis (1). Differential gene expression has been described for many bacteria under various growth conditions or during infection (4 – 8). Due to the difficulty in studying bacterial gene expression in vivo, many studies have been performed in in vitro systems meant to mimic the host environment, including iron and nutrient limitation, conditions of low oxygen, biofilm versus planktonic bacterial growth, and following exposure to blood in vitro (4 – 8). These studies have provided valuable information about genes involved in the bacterial response to particular host environments when mimicked in vitro; however, the in vivo setting is far more complex. In addition to in vitro studies, S. aureus gene expression has been characterized in vivo. Expression of a limited number of genes has been investigated in both device-related and wound infections (9 – 11), during skin infections (12, 13), human nasal colonization (14), kidney infection in mice (13), and in a cotton rat nasal colonization model (15). In these studies, transcript levels in vivo were compared with transcript levels measured under in vitro growth conditions. A comparison of gene expression in S. aureus between disease states has not been reported. The goal of this study was to compare the expression profile of 23 putative S. aureus virulence determinants in two clinical isolates during three stages of infection: nasal colonization, early bacteremia, and infected heart tissue (thromboembolic lesions) in a sepsis model. These models were chosen to represent the stepwise progression of individual S. aureus strains from a commensal (asymptomatic nasal colonization) to a pathogen (from early bacteremia and finally to an invasive infection during sepsis). Our results revealed upregulation of sdrC, fnbA, fhuD, sstD, and hla in S. aureus as an invasive pathogen compared to a commensal. These findings suggest roles for these proteins in the progression of S. aureus infection and support further investigation of these factors as therapeutic targets. RESULTS Quantitative real-time PCR (qRT-PCR) was utilized to determine how S. aureus gene expression responds to environmental changes in the host, using different animal models (cotton rat nasal colonization and murine bacteremia and lethal sepsis). Cotton rat nasal colonization was chosen because it is well characterized and closely resembles chronic human nasal colonization. Alternatively, S. aureus is either cleared or requires antibiotics for chronic murine nasal colonization (16 – 20). Murine bacteremia and sepsis models were used because these models are not established for cotton rats. Four S. aureus gene classes were chosen for analysis: protein and carbohydrate adhesins, metal cation transporters, exotoxins, and immune evasion proteins (see Table S1 in the supplemental material). One concern when running qRT-PCR on samples purified from an in vivo setting is that contaminants could copurify with the cDNA that inhibit the PCR. To address this concern, cDNA samples from each of the different infection sites (cotton rat nose and mouse blood and heart) were spiked into a control PCR assay specific for Pseudomonas aeruginosa pcrV on a plasmid. All of the samples amplified the pcrV gene with similar efficiency, indicating that none of the in vivo purified cDNA samples regardless of infection site adversely affected PCR efficiency (see Fig. S1 in the supplemental material). Nasal colonization. Experiments were performed with two S. aureus clinical isolates, SF8300 (CC8; USA300 methicillin-resistant S. aureus [MRSA]) and ARC633 (CC15; methicillin-susceptible S. aureus [MSSA]) obtained from a skin abscess and from a nasal carrier, respectively. To establish a baseline condition (commensal), transcript levels were measured during cotton rat nasal colonization. To avoid the pitfalls of comparing expression data to data for expression levels under an arbitrary in vitro growth condition, expression levels were represented as the fold change from the minimum and maximum in vitro expression levels (a description of the in vitro growth conditions that resulted in these expression levels can be found in Table S2 in the supplemental material). 16S rRNA was selected as an internal control for normalization because its expression varied less than 2-fold for both SF8300 and ARC633 under various in vitro growth conditions (e.g., early-log, mid-log, late-log, and stationary phases in trypticase soy broth [TSB], RPMI, and human serum) (see Fig. S2 in the supplemental material). As such, a change in gene expression was defined as a greater-than-2-fold increase or decrease in transcript levels. During nasal colonization, transcript levels of four adhesin genes (clfB, sdrC, sdrD, and tarK) increased over the highest observed in vitro expression levels in both strains. An additional four genes exhibited increased expression over the highest in vitro expression levels in one strain (clfA, icaB, efb, and sasG) (Fig. 1). Increased clfB, sdrC, sdrD, sasG, and tarK transcript levels support previous results implicating their corresponding proteins in nasal colonization (14, 15, 21 – 23). Metal ions are limiting in vivo; therefore, one would expect metal cation transporters to be upregulated (24, 25). As expected, most metal cation transporter genes were upregulated relative to the minimal in vitro expression level (Fig. 2). The genes encoding SpA and Sbi showed increased expression over their lowest in vitro expression level and were unchanged or decreased from the highest in vitro expression level. Toxin gene expression decreased compared to the lowest in vitro expression condition (Fig. 3). Of note, ARC633 does not encode lukF-PV. FIG 1  Protein and carbohydrate adhesin genes in a cotton rat nasal colonization model. Transcripts were assessed relative to 16S rRNA, and results from nasal colonization were compared to the minimum expression in vitro (A) and the maximum expression in vitro (B). In vitro growth conditions are described in Table S2 in the supplemental material. Analysis was performed for two S. aureus strains, SF8300 (black bars) and ARC633 (blue bars). These results are the means of three independent experiments with 12 animals/experiment. *, P  < 0.05 (Student’s t test). clfB expression was not detected in vitro, and so calculations for clfB expression were based on an in vitro threshold cycle CT value equal to the limit of detection (40 cycles). FIG 2  Metal cation acquisition gene expression in a cotton rat nasal colonization model. Transcripts were assessed relative to 16S rRNA, and results from nasal colonization were compared to the minimum expression level in vitro (A) and the maximum expression level in vitro (B). In vitro growth conditions are described in Table S2 in the supplemental material. Analysis was performed for two S. aureus strains, SF8300 (black bars) and ARC633 (blue bars). These results are the means of three independent experiments with 12 animals/experiment. *, P  < 0.05 (Student’s t test). FIG 3  Immune evasion and exotoxin gene expression in a cotton rat nasal colonization model. Transcripts were assessed relative to 16S rRNA, and results from nasal colonization were compared to the minimum expression level in vitro (A) and the maximum expression level in vitro (B). In vitro growth conditions are described in Table S2 in the supplemental material. Analysis was performed for two S. aureus strains, SF8300 (black bars) and ARC633 (blue bars). These results are the means of three independent experiments with 12 animals/experiment. *, P < 0.05 (Student’s t test). Nasal colonization versus early bacteremia. Gene expression was then assessed in the blood of bacteremic mice 1 h postinfection (via intravenous [i.v.] injection). Transcript levels were compared to those observed in nasal colonization to determine which genes the bacteria modulate upon exposure to the bloodstream. Of the adhesins, only fnbA increased expression in both strains compared to nasal colonization, whereas sdrC and icaB showed increased expression in SF8300 and sasA, tagO, efb, and ebpS showed increased expression in ARC633 upon exposure to the bloodstream. Three metal cation acquisition genes had increased expression (mntC, fhuD, and sstD) compared to nasal colonization in ARC633. The hla gene was upregulated in both strains, whereas lukF-PV (SF8300) and sbi and spa (ARC633) were upregulated in one strain (Fig. 4). Taken together, these results indicate that individual S. aureus isolates respond differently to environmental change and may use different virulence determinants during the same type of infection. FIG 4  Differential gene expression between nasal colonization and early bacteremia. Transcripts were assessed relative to 16S rRNA, and results from the bloodstream were compared to the expression results from nasal colonization for protein and carbohydrate adhesins (A), metal cation acquisition genes (B), and immune evasion genes and exotoxins (C). Analysis was performed for two S. aureus strains, SF8300 (black bars) and ARC633 (blue bars). These results are the means of three independent experiments with 10 animals/experiment. *, P < 0.05 (Student’s t test). clfA, clfB, and sasG expression was not detected in the blood samples, and so calculations for clfA, clfB, and sasG expression were based on the threshold cycle (CT ) value equal to the limit of detection (40 cycles). Transition from early bacteremia to heart lesions. Transcript levels were measured in bacteria present in saline-perfused hearts 14 h postchallenge (i.v.) and compared to those in blood 1 h postinfection. The hearts were flushed with saline to ensure transcript levels were measured from bacteria in the heart tissue and not in residual blood. It should be noted that, similar to previous reports (26), we did observe histological evidence of thromboembolic lesions in the hearts of infected mice (data not shown). The comparisons performed here were undertaken to highlight genes regulated upon transition to the thromboembolic heart lesions characteristic of S. aureus sepsis (26), relative to the initial stages of bacteremia. Six adhesin genes (clfA, sdrC, sasF, tagO, fnbA, and tarK) displayed increased expression in both strains, and sasA (SF8300) and icaB (ARC633) showed increased expression in one strain in thromboembolic lesions relative to early bacteremic blood. Of these, clfA, tagO, and fnbA have been implicated in establishing organ colonization (26 – 29). Four cation transport genes (mntC, isdB, fhuD, and isdA) exhibited increased expression in both strains relative to early bacteremic blood, whereas isdH and sstD showed increased expression in ARC633. Upon seeding of the heart, hla, sbi, and lukF-PV all showed increased expression in SF8300, while hla and sbi remained constant in ARC633 (Fig. 5). In general, these results suggest that S. aureus utilizes multiple adhesins in the process of bloodstream escape and organ invasion. Additionally, upregulation of the metal cation transporters points to the iron-limiting nature of the heart, while exotoxins and immune evasion genes are likely utilized to escape the bloodstream and host defenses. FIG 5  Differential gene expression in the transition from the bloodstream to heart tissue. Transcripts were assessed relative to the 16S rRNA, and results from the heart tissue were compared to the expression results from the bloodstream for protein and carbohydrate adhesins (A), metal cation acquisition genes (B), and immune evasion genes and exotoxins (C). Analysis was performed for two S. aureus strains, SF8300 (black bars) and ARC633 (blue bars). These results are the means of three independent experiments with 10 animals/experiment. *, P < 0.05 (Student’s t test). clfB and sasG expression was not detected in the blood samples or the heart samples; therefore, expression numbers are not reported for these genes. Isogenic mutant analysis. To compare gene expression analysis with a gene’s role in pathogenesis, isogenic deletion mutants were constructed in SF8300. The genes (clfA, clfB, sdrC, isdH, isdB, hla, and spa) were chosen to represent a variety of expression profiles in the three models. The adhesins clfA and sdrC were chosen based on their consistent upregulation during infection. clfB, isdH, and isdB were chosen based on their upregulation in the nasal colonization model and in the transition from bloodstream to invasive pathogen (isdB). The remaining genes (hla and spa) showed substantially increased expression in two models and are known to play a role in S. aureus pathogenesis. Cotton rats were infected intranasally with isogenic mutants, and CFU were compared to exposuure to wild type (WT) 4 days postinfection (Fig. 6). Only the ΔsdrC mutant showed a trend toward a reduction (~0.5 log) in nasal colonization, and this trend was not significant. When both sdrC and clfB (an adhesin shown to play a role in nasal colonization [21, 30]) were deleted (ΔsdrC ΔclfB), a significant reduction in CFU in the nares was observed. FIG 6  CFU enumeration of SF8300 wild-type and mutant strains in a cotton rat nasal colonization model. Groups of 12 cotton rats were challenged intranasally with 5 × 105 CFU S. aureus in 10 µl (5 µl per nostril). For ΔsdrC ΔclfB, P =0.0047 (Mann-Whitney test). Data shown are from a single replicate of three independent experiments. Mutant strains were compared to WT for a change in heart CFU following i.v. infection. Knockout mutants for clfA, sdrC, isdB, and hla all resulted in decreased heart CFU, consistent with increased expression observed in thromboembolic lesions (Fig. 7). Knockout mutants of genes that were downregulated or unchanged in thromboembolic lesions did not impact CFU recovery from the heart, with the exception of ΔspA (Fig. 7). It should be noted that all isogenic mutants were determined to have no growth defects in TSB compared to WT SF8300 (data not shown). FIG 7  CFU enumeration of SF8300 wild-type and mutant strains in a murine thromboembolic lesion model. Groups of 10 female BALB/c mice were challenged intravenously (i.v.) with 200 µl of S. aureus (5 × 107 CFU) by tail vein injection. P values were as follows: Δhla, 0.0030; ΔclfA, <0.0001; ΔsdrC, 0.0050; ΔisdB, <0.0001 (Mann-Whitney test). Data shown are from a single replicate of three independent experiments. DISCUSSION Understanding how S. aureus regulates gene expression as it transitions from colonization to infection is critical to understanding how S. aureus causes disease. We used qRT-PCR to examine transcript levels of known or putative virulence determinants in bacteria isolated from relevant animal models. In vivo bacterial gene expression data are typically presented relative to an arbitrary in vitro condition, making interpretation of gene expression data in disease states difficult. To avoid this limitation, gene expression patterns were compared directly from nasal colonization to bacteremia to thromboembolic lesions. This reduced the need to compare expression to an in vitro condition and allowed for the assessment of gene expression changes in the bacterium at different stages of infection (e.g., commensal to pathogenic). Out of necessity, nasal colonization expression was compared to in vitro expression to establish a baseline for future comparisons. Given the well-documented role of adhesins during nasal colonization (14 – 17, 21 – 23, 30, 31), it was not surprising that several adhesin genes were dramatically upregulated (5- to 21,000-fold) in at least one strain relative to their highest expression in vitro. For example, ClfB, SdrC, SdrD, and SasG have been proposed to play roles in nasal colonization, in either in vivo (21) or in vitro (22, 23) studies. A central role for SdrC and ClfB in nasal colonization was confirmed, as the ΔsdrC ΔclfB double mutant showed a significant CFU reduction relative to wild-type SF8300. To our knowledge, this is the first direct evidence of a role for SdrC in nasal colonization in vivo. SdrC has been reported to bind β-neurexin, a protein found primarily in brain tissue, not on nasal epithelium (32), suggesting an alternate ligand for SdrC. In addition to clfB and sdrC, transcript levels of clfA, icaB, sdrD, tarK, sasG, and efb were increased, suggesting that they also play a role in nasal colonization. These results support the hypothesis that nasal colonization is a multifactorial process requiring numerous adhesins (23). Nearly all metal cation acquisition genes analyzed during nasal colonization showed decreased expression relative to their highest in vitro expression, although expression was increased above their lowest in vitro level. This was not unexpected, as the nares are a low-iron environment (33), but their environment may not be as metal ion restricted as RPMI medium. Although isdH expression exhibited a slight increase (3-fold) during nasal colonization compared to the maximum in vitro expression, there was no change in bacterial CFU in the nares of cotton rats colonized with ΔisdH versus WT. This does not rule out a role for IsdH in iron acquisition, but it could reflect the redundant nature of S. aureus iron acquisition systems (33). Similar to published results, hla expression was low in the nares (14), and the Δhla mutant did not affect bacterial CFU in the nares, suggesting hla does not play a significant role in nasal colonization. However, due to the multifactorial nature of nasal colonization, a role for the toxins cannot be ruled out. Finally, the immune evasion proteins exhibited a large increase in expression in nasal colonization. This was expected, as this is the bacterium’s first encounter with the immune system. Several adhesin genes (fnbA, sdrC, iCab, sasA, tagO, efb, and ebpS), all of the immune evasion genes, and both toxins increased in expression at least 2-fold in the bloodstream of mice relative to nasal colonization, while the metal cation acquisition genes were largely unchanged or decreased relative to expression in the nares. Increased adhesin gene expression suggests that, upon exposure to the bloodstream, S. aureus begins to adhere to host cells or tissue. While a role for icaB during bacteremia has been reported (34), sdrC, sasA, tagO, efb, and ebpS have not been reported to play a role during bacteremia. Several of these adhesins have been studied in both in vitro and in vivo systems, and those results, combined with the results presented here, provide insights into a role for these genes in S. aureus pathogenesis. For example, FnbA and TagO have been reported to play a role in adherence to endothelial cells (29, 35, 36). Although SasA has not been implicated in bloodstream infection, it has been reported that SasA antibodies are detected in convalescent patient sera, providing further evidence that SasA is expressed by S. aureus during systemic infection (37). These data suggest S. aureus, even early in bacteremia, rapidly expresses adhesins to facilitate binding to endothelial surfaces, presumably to escape the bloodstream and colonize host tissues. The three other upregulated adhesin genes, efb, ebpS, and sdrC, have not been implicated in S. aureus bacteremia; however, our data suggest a role for these genes in S. aureus bacteremia and/or endothelial adherence. The metal cation acquisition proteins (except for isdA and isdB) were still highly expressed in the bloodstream, where they aid in scavenging iron. The difference in isdB and isdA expression relative to nasal colonization could highlight differences in nutrient availability and host iron-scavenging proteins in the nares versus the blood (38). Increased expression of sbi, spA, and lukF-PV suggests that the bacterium is actively engaged in evading the host immune system upon introduction into the bloodstream. Increased hla expression during bacteremia supports previous studies describing a role for alpha-toxin (AT) in bloodstream infections (39). Transition from bacteremia to heart tissue led to drastically increased expression of most adhesin genes and all metal cation acquisition, toxin, and immune evasion genes, with the exception of spa, relative to bloodstream expression. Increased adhesin gene expression may be expected, as these proteins are generally thought to facilitate adherence to and colonization of host tissues (40, 41). Although some of the upregulated adhesins have been shown to play a role in establishing organ lesions (ClfA [26, 27], FnbA [35], and TagO [29]), many of them do not have a reported role in organ lesion formation. Increased expression of sdrC (13-fold), sasA (4-fold), and sasF (11-fold) are of particular interest in that little is known about their roles in S. aureus virulence. While a ΔsdrC mutant strain led to a relative decrease in CFU recovered from heart lesions compared to WT, further experiments are required to elucidate the role of these putative adhesins in severe S. aureus infections. The upregulation of all metal ion acquisition genes (with the exception of isdH and sstD in strain SF8300) indicates that heart lesions may be more iron limiting than blood. Previous studies have shown iron content varies among organs, with heart muscle containing less iron than other organs, although free blood iron levels were not determined in those studies (42). The expression of spa in the heart is decreased relative to that in the bloodstream; however, Δspa resulted in decreased heart CFU relative to infection with WT. This may be due to high spa expression in the bloodstream; therefore, a decrease in spa between the bloodstream and the heart still results in a significant amount of protein A present on the bacterial surface. Alternatively, perhaps Δspa reduces bacterial survival in the blood, resulting in lower bacterial numbers invading the heart tissue. Although AT has been shown to be important in establishing S. aureus infections, including infective endocarditis (43), there is no evidence that it plays a role in thromboembolic lesions. The data presented here suggest AT plays a role in infecting the heart tissue, both through increased transcript levels and decreased CFU recovered from heart tissue following infection with Δhla relative to infection with WT. One hypothesis for AT’s role in establishing the heart lesions involves its activation of ADAM10-mediated E-cadherin cleavage, resulting in vascular leakage and bacterial invasion of heart tissue (44). To our knowledge, these results are the first example of a study designed to directly compare virulence factor expression by a bacterium during commensal and pathogenic states. Additionally, this study has shown that this type of analysis is possible, and valuable information can be garnered by comparing expression patterns between colonization and disease states, laying the groundwork for large-scale microarray studies for a broader view of S. aureus as a commensal and a pathogen. Recently, a microarray study examining S. aureus isolated from human skin infections was performed and, as expected, many of the upregulated genes correlated well with our in vivo results (e.g., hla, isdB, isdA, and sitC) (13). The results obtained in this study support previous reports describing the involvement of individual S. aureus genes and proteins in the three disease states, and they provide new information about genes which may play a role in nasal colonization (icaB, clfA, and efb), early bloodstream infection (sdrC, sasA, tagO, fnbA, efb, ebpS, mntC, fhuD, and sstD), and thromboembolic lesions (sdrC, sasA, sasF, isdH, mntC, sstD, fhuD, spA, sbi, and hla). The steadily increasing expression of sdrC, fnbA, fhuD, sstD, and hla (in at least one strain) from nasal colonization to bacteremia to heart lesions suggests a role for these genes in the transition from commensal to pathogen. The expression patterns of the chosen genes often varied between the strains tested, highlighting S. aureus diversity and stressing the need to evaluate multiple clinical isolates when identifying important virulence factors. The information collected provides a basic understanding of S. aureus disease pathogenesis and how a commensal becomes a pathogen, and this understanding may ultimately lead to the development of targeted therapeutics. MATERIALS AND METHODS S. aureus strains, media, and growth. SF830 is a prototypical USA300-0114 community-associated methicillin-resistant S. aureus strain (45). ARC633 (CC15; MSSA) is a nasal colonization isolate provided by David A. Bruckner, UCLA Medical Center. Bacteria were cultured with shaking (250 rpm) at 37°C in TSB for growth curves and for in vitro gene expression cultures. For RNA purification from in vitro cultures, samples were mixed 1:2 with RNAprotect (Qiagen), incubated at room temperature for 5 min, and pelleted by centrifugation. The pellet was resuspended in 1 ml of TRIzol (Invitrogen). The TRIzol-treated samples were lysed by using a FastPrep 24 homogenizer with lysing matrix B tubes (MP Biomedicals). RNA purification was performed as described below. Animal challenge stocks were prepared by growing bacteria overnight in TSB, then diluting cultures into fresh TSB and growing until the optical density at 600 nm reached ~0.8. The bacteria were collected by centrifugation and resuspended in phosphate-buffered saline (PBS; Invitrogen) plus 10% glycerol and frozen at −80°C in aliquots for use in all animal studies. Cotton rat nasal colonization model. Cotton rats (n = 12) were challenged intranasally with 5 × 105 CFU S. aureus in 10 µl (5 µl per nostril). Four days postchallenge, the nares were harvested and placed into 20 ml of RNAprotect (for RT-PCR) and incubated for 5 min, or placed into 2 ml of PBS plus 0.1% Tween 20 (for CFU enumeration). For RT-PCR, nares were placed into 10 ml of TRIzol (Invitrogen) and vigorously vortexed to remove bacteria. The TRIzol-treated samples were lysed using the FastPrep 24 homogenizer with lysing matrix B tubes (MP Biomedicals). RNA purification was performed as described below. All RT-PCR data are reported as means of 3 independent experiments using 12 animals/experiment. For CFU enumeration, nares were homogenized using a Polytron PT-10-35 GT apparatus. The samples were serially diluted and plated onto Staph Chrome agar plates (BD Biosciences). All animal use protocols were reviewed and approved by MedImmune’s IACUC and complied with the animal welfare standards of the USDA, Guide for the Care and Use of Laboratory Animals, and AAALAC International. Murine bacteremia. Female BALB/c mice (n = 10) were challenged by tail vein injection of 1 × 108 CFU of S. aureus (200 µl). One hour postchallenge, blood was collected from and pooled in 20 ml of RNAprotect. The bacteria were pelleted by centrifugation, resuspended in 1 ml of TRIzol, and treated as described above. All RT-PCR data are reported as means of 3 independent experiments using 10 animals/experiment. Murine sepsis. Female BALB/c mice (n = 10) were challenged by tail vein injection of 1 × 107 CFU S. aureus (200 µl). Fourteen hours postchallenge, hearts were harvested and placed into 20 ml of RNAprotect (for RT-PCR) and incubated for 5 min, or tissue was placed in 1 ml of PBS plus 0.1% Tween 20 (for CFU enumeration). Bacteria and heart tissue were lysed, and RNA was purified as described above. All RT-PCR data are reported as means of 3 independent experiments using 10 animals/experiment. For CFU enumerations, hearts were homogenized in 1 ml of PBS plus 0.1% Tween 20 in lysing matrix A tubes (MP Biomedicals) in a FastPrep 24 homogenizer. Samples were serially diluted and plated onto trypticase soy agar plates (BD Biosciences). Construction of in-frame gene deletions. In-frame deletions of selected genes were constructed by allelic replacement using pKOR1 (46). Primers X1-X2 (see Table S3 in the supplemental material) were used to amplify approximately 1,000 bp that corresponded to the first 84 to 153 nucleotides from the start codon and flanking 5′ region. Primers X3-X4 were used to amplify approximately 1,000 bp that corresponded to the last 33 to 159 nucleotides from the stop codon and flanking 3′ region (see Table S3). X1-X2 and X3-X4 PCR products were spliced together by overlap PCR using primers X5 and X6. Attachment sites (attB), appended to 5′ ends of primers X5 and X6 were recombined with the attP sequences flanking a lambda recombination cassette on pKOR1 in the presence of bacteriophage lambda integrase and Escherichia coli integration host factor (Clonase; Invitrogen) and electroporated into E. coli. The in-frame deletion constructs were electroporated into S. aureus RN4220 and then transduced with Φ11 into SF8300. Allelic replacement was performed as described elsewhere (46). Allelic replacement mutants were identified by PCR and DNA sequencing using primers X1 to X4 and primers X1, X4, S1, and S2, respectively (see Table S3). Multiple gene deletions were carried out sequentially. RNA preparation. Chloroform (200 µl) was added to 1 ml of lysed bacteria and centrifuged at 14,000 × g, and the top clear layer was removed, added to 1 ml of isopropanol, and then incubated at −20°C overnight. RNA was pelleted by centrifugation (14,000 × g) for 30 min. The pellet was washed with 70% ethanol, dried, resuspended in 100 µl of RNase-free water, and digested with RNase-free DNase (Promega) for 1 h. Following digestion, 1 ml of TRIzol and 200 µl of chloroform were added. RNA precipitation and DNase digestion procedures were repeated. RNA was then purified using the RNeasy minikit (Qiagen). RT-PCR. RNA was reverse transcribed into cDNA by using the SuperScript III cDNA synthesis kit (Invitrogen). TaqMan primers (see Table S4 in the supplemental material) containing a 6-carboxyfluorescein reporter and nonfluorescent quencher were designed using the TaqMan design tool (Life Technologies). cDNA samples were assayed in triplicate using 16S rRNA as a control. Samples were assayed using TaqMan universal PCR master mix (Applied Biosystems) on an Applied Biosystems 7900HT apparatus with standard cycling protocols and analyzed using SDS software. Relative expression values were calculated using the ΔΔCT method with 16S RNA as the normalizer (47 – 49). 16S RNA was determined to be the most stable housekeeping gene in our experiments, based on expression under a variety of in vitro growth conditions (rich medium, minimal medium, or in the presence of serum). SUPPLEMENTAL MATERIAL Figure S1  Impact of cDNA from different RNA source tissues on the efficiency of amplification of a test amplicon. Download Figure S1, DOCX file, 0.02 MB Figure S2  16S rRNA levels in S. aureus strains SF8300 and ARC633 under various in vitro growth conditions. Download Figure S2, DOCX file, 0.1 MB Table S1  Genes analyzed in this study Table S1, DOC file, 0.1 MB. Table S2  Growth conditions for the highest and lowest in vitro expression levels Table S2, DOC file, 0.05 MB. Table S3  Primers used for in-frame gene deletions using pKOR1 Table S3, DOCX file, 0.01 MB. Table S4  Primers and probes used for TaqMan qPCR Table S4, DOCX file, 0.01 MB.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, CA USA )
                1553-7366
                1553-7374
                26 May 2016
                May 2016
                : 12
                : 5
                : e1005519
                Affiliations
                [001]Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, United States of America
                University of Wisconsin Medical School, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                Article
                PPATHOGENS-D-16-00270
                10.1371/journal.ppat.1005519
                4881934
                27227827
                de6c379c-0d72-4ec0-b1d6-e56fbe64ba98
                © 2016 McCall et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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                Funding
                LIM acknowledges receiving a postdoctoral fellowship from the Canadian Institutes of Health Research (338511, http://www.cihr-irsc.gc.ca/). This work was supported in part by the European Union Seventh Framework Programme (602773-KINDRED to JHM, http://cordis.europa.eu/fp7/home_en.html).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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