Exoproteome of <i>Staphylococcus aureus</i> Reveals Putative Determinants of Nasal Carriage

Due to the increasing prevalence of nosocomial and community-acquired antibiotic resistant Staphylococcus aureus (SA), understanding the determinants of SA nasal carriage has become a major imperative. Previous research has revealed many host and bacterial factors that contribute to SA nasal carriage. To assess bacterial factors that facilitate nasal carriage, we compared the exoproteome of a nasal carrier strain of SA to a genetically similar noncarrier strain. Additionally, the carrier strain biofilm exoproteome was also compared against its planktonic counterpart. Using high throughput proteomics, it was observed that the carrier strain of SA secretes a greater number of proteins that may promote successful colonization of the human nose, including cell attachment and immunoevasive proteins, than the noncarrier strain. Similarly, SA carrier strain biofilm exoproteome contains a greater number of immunoevasive proteins than its planktonic counterpart. Analysis of the most abundant immunoevasive proteins revealed that Staphylococcal protein A was present at significantly higher levels in carrier than in noncarrier strains of SA, suggesting an association with nasal carriage. While further analyses of specific differences between carrier and noncarrier strains of SA are required, many of the differentially expressed proteins identified can be considered to be putative determinants of nasal carriage

Exoproteome of <i>Staphylococcus aureus</i> Reveals Putative Determinants of Nasal Carriage

Due to the increasing prevalence of nosocomial and community-acquired antibiotic resistant Staphylococcus aureus (SA), understanding the determinants of SA nasal carriage has become a major imperative. Previous research has revealed many host and bacterial factors that contribute to SA nasal carriage. To assess bacterial factors that facilitate nasal carriage, we compared the exoproteome of a nasal carrier strain of SA to a genetically similar noncarrier strain. Additionally, the carrier strain biofilm exoproteome was also compared against its planktonic counterpart. Using high throughput proteomics, it was observed that the carrier strain of SA secretes a greater number of proteins that may promote successful colonization of the human nose, including cell attachment and immunoevasive proteins, than the noncarrier strain. Similarly, SA carrier strain biofilm exoproteome contains a greater number of immunoevasive proteins than its planktonic counterpart. Analysis of the most abundant immunoevasive proteins revealed that Staphylococcal protein A was present at significantly higher levels in carrier than in noncarrier strains of SA, suggesting an association with nasal carriage. While further analyses of specific differences between carrier and noncarrier strains of SA are required, many of the differentially expressed proteins identified can be considered to be putative determinants of nasal carriage

Exoproteome of <i>Staphylococcus aureus</i> Reveals Putative Determinants of Nasal Carriage

Due to the increasing prevalence of nosocomial and community-acquired antibiotic resistant Staphylococcus aureus (SA), understanding the determinants of SA nasal carriage has become a major imperative. Previous research has revealed many host and bacterial factors that contribute to SA nasal carriage. To assess bacterial factors that facilitate nasal carriage, we compared the exoproteome of a nasal carrier strain of SA to a genetically similar noncarrier strain. Additionally, the carrier strain biofilm exoproteome was also compared against its planktonic counterpart. Using high throughput proteomics, it was observed that the carrier strain of SA secretes a greater number of proteins that may promote successful colonization of the human nose, including cell attachment and immunoevasive proteins, than the noncarrier strain. Similarly, SA carrier strain biofilm exoproteome contains a greater number of immunoevasive proteins than its planktonic counterpart. Analysis of the most abundant immunoevasive proteins revealed that Staphylococcal protein A was present at significantly higher levels in carrier than in noncarrier strains of SA, suggesting an association with nasal carriage. While further analyses of specific differences between carrier and noncarrier strains of SA are required, many of the differentially expressed proteins identified can be considered to be putative determinants of nasal carriage

Exoproteome of <i>Staphylococcus aureus</i> Reveals Putative Determinants of Nasal Carriage

Due to the increasing prevalence of nosocomial and community-acquired antibiotic resistant Staphylococcus aureus (SA), understanding the determinants of SA nasal carriage has become a major imperative. Previous research has revealed many host and bacterial factors that contribute to SA nasal carriage. To assess bacterial factors that facilitate nasal carriage, we compared the exoproteome of a nasal carrier strain of SA to a genetically similar noncarrier strain. Additionally, the carrier strain biofilm exoproteome was also compared against its planktonic counterpart. Using high throughput proteomics, it was observed that the carrier strain of SA secretes a greater number of proteins that may promote successful colonization of the human nose, including cell attachment and immunoevasive proteins, than the noncarrier strain. Similarly, SA carrier strain biofilm exoproteome contains a greater number of immunoevasive proteins than its planktonic counterpart. Analysis of the most abundant immunoevasive proteins revealed that Staphylococcal protein A was present at significantly higher levels in carrier than in noncarrier strains of SA, suggesting an association with nasal carriage. While further analyses of specific differences between carrier and noncarrier strains of SA are required, many of the differentially expressed proteins identified can be considered to be putative determinants of nasal carriage

BVAB Induce an Innate Cytokine Response in Female Reproductive Epithelia.

<p>Transwell monolayers of each epithelial line were inoculated with indicated bacteria (Lact = <i>L. johnsonii</i>, average MOI = 33, Gard = <i>G. vaginalis</i>, average MOI = 15, Ato = <i>A. vaginae,</i> average MOI = 13) and 24 hr post-inoculation conditioned media were analyzed by multiplex cytokine bead array. Cytokine values are averaged from three independent experiments. Relative induction, represented here as fold expression and shaded accordingly, was calculated as the average increase in cytokine concentration compared to mock-inoculated control cells from three independent experiments. Significant differences in cytokine concentrations between bacteria-inoculated and mock condition are indicated by one (p<0.05), two (p<0.01) or three (p<0.001) asterisks.</p

Human β-Defensin Gene Expression Is Upregulated in Reproductive Epithelia in Response to BVAB.

<p>Confluent monolayers of epithelial cells were inoculated with bacteria and coincubated for 48 hr, then analyzed by RTqPCR for hBD2 and hBD3 expression. Transcript expression is reported relative to 0 hr cells, and is shown as the average of three independent experiments. Average MOI are: 10 for <i>L. johnsonii</i>, 5.8 for <i>G. vaginalis</i>, and 5.6 for <i>A. vaginae</i>. Asterisks indicate a significant (p<0.05) increase over the <i>L. johnsonii</i>-treated condition.</p

Identification and Characterization of Bacterial Vaginosis-Associated Pathogens Using a Comprehensive Cervical-Vaginal Epithelial Coculture Assay

<div><p>Bacterial vaginosis (BV) is the most commonly treated female reproductive tract affliction, characterized by the displacement of healthy lactobacilli by an overgrowth of pathogenic bacteria. BV can contribute to pathogenic inflammation, preterm birth, and susceptibility to sexually transmitted infections. As the bacteria responsible for BV pathogenicity and their interactions with host immunity are not understood, we sought to evaluate the effects of BV-associated bacteria on reproductive epithelia. Here we have characterized the interaction between BV-associated bacteria and the female reproductive tract by measuring cytokine and defensin induction in three types of FRT epithelial cells following bacterial inoculation. Four BV-associated bacteria were evaluated alongside six lactobacilli for a comparative assessment. While responses differed between epithelial cell types, our model showed good agreement with clinical BV trends. We observed a distinct cytokine and human β-defensin 2 response to BV-associated bacteria, especially <em>Atopobium vaginae</em>, compared to most lactobacilli. One lactobacillus species, <em>Lactobacillus vaginalis</em>, induced an immune response similar to that elicited by BV-associated bacteria, stimulating significantly higher levels of cytokines and human β-defensin 2 than other lactobacilli. These data provide an important prioritization of BV-associated bacteria and support further characterization of reproductive bacteria and their interactions with host epithelia. Additionally, they demonstrate the distinct immune response potentials of epithelial cells from different locations along the female reproductive tract.</p> </div

<i>L. vaginalis</i> Induces a Greater Immune Response than Other Vaginal Lactobacilli.

<p>Confluent monolayers of reproductive epithelia were inoculated with commensal lactobacilli or BVAB, and after 24 hr conditioned media was analyzed for IL-6, IL-8 and hBD2 protein. BVAB are filled black bars, <i>L. vaginalis</i> is hatched, and all other lactobacilli are white bars. MOI are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050106#pone-0050106-g005" target="_blank">Figure 5</a>. Protein is shown as fold expression compared to a mock-treated condition, and one, two, or three asterisks indicate values that are significantly (p<0.05, p<0.01, or p<0.001, respectively) different from the <i>L. vaginalis</i>-treated condition.</p

<i>A. vaginae</i> Induces Epithelial Expression of Soluble and Cell-Associated hBD2, a Protein That Attracts Primary Lymphocytes.

<p>A) Confluent monolayers of epithelial cells were inoculated with bacteria. Twenty-four hr post-inoculation, conditioned media were collected, clarified, and analyzed by ELISA to quantify concentrations of soluble hBD2 protein. Average MOI are: 5.8 for <i>L. johnsonii</i>, 2.5 for <i>G. vaginalis</i>, and 1.4 for <i>A. vaginae</i>. Results are averaged from 3 independent experiments, and asterisks indicate significant (p<0.001) increase over mock<i>-</i>treated condition. B) Confluent monolayers were inoculated with <i>A. vaginae</i>, or mock-inoculated. 24, 48, and 72 hr post-inoculation, cell monolayers were acid-extracted and analyzed by AU-PAGE western to quantify cell-associated hBD2 protein. A recombinant hBD2 protein standard (shown in ng per lane) was run alongside cell extracts for semi-quantitative comparison. Average MOI is 21. Shown is one example of three independent experiments. C) Unstimulated primary lymphocytes were isolated and evaluated for chemotaxis toward recombinant hBD2 protein. Chemotactic index is the ratio of migrated cells in hBD2-containing wells over vehicle-control wells, and significant increases over the matched vehicle condition are shown by three (p<0.001) asterisks.</p

<i>L. vaginalis</i> Elicits hBD2 Gene Induction from Reproductive Epithelia.

<p>Confluent monolayers of epithelial cells were inoculated with bacteria and coincubated for up to 24 hr, then analyzed by RTqPCR for hBD2 and hBD3 expression. Transcript expression was normalized to mock-inoculated cells, and is shown as the average of three or four independent experiments. Average MOI are: 6.9 for <i>L. acidophilus,</i> 7.3 for <i>L. crispatus,</i> 9.7 for <i>L. gasseri,</i> 7.3 for <i>L. jensenii,</i> 7.3 for <i>L. johnsonii,</i> 3.4 for <i>L. vaginalis,</i> 3.7 for <i>A. vaginae,</i> 3.1 for <i>G. vaginalis,</i> 3.6 for <i>M. curtisii,</i> and 3.9 for <i>P. bivia</i>. Asterisks indicate a significant (p<0.05) increase in expression over mock-treated cells for at least one timepoint.</p