MNPmApp: An image analysis tool to quantify mononuclear phagocyte distribution in mucosal tissues.

Mononuclear phagocytes (MNPs) such as dendritic cells and macrophages perform key sentinel functions in mucosal tissues and are responsible for inducing and maintaining adaptive immune responses to mucosal pathogens. Positioning of MNPs at the epithelial interface facilitates their access to luminally-derived antigens and regulates MNP function through soluble mediators or surface receptor interactions. Therefore, accurately quantifying the distribution of MNPs within mucosal tissues as well as their spatial relationship with other cells is important to infer functional cellular interactions in health and disease. In this study, we developed and validated a MATLAB-based tissue cytometry platform, termed "MNP mapping application" (MNPmApp), that performs high throughput analyses of MNP density and distribution in the gastrointestinal mucosa based on digital multicolor fluorescence microscopy images and that integrates a Monte Carlo modeling feature to assess randomness of MNP distribution. MNPmApp identified MNPs in tissue sections of the human gastric mucosa with 98 ± 2% specificity and 76 ± 15% sensitivity for HLA-DR+ MNPs and 98 ± 1% specificity and 85 ± 12% sensitivity for CD11c+ MNPs. Monte Carlo modeling revealed that mean MNP-MNP distances for both HLA-DR+ and CD11c+ MNPs were significantly lower than anticipated based on random cell placement, whereas MNP-epithelial distances were similar to randomly placed cells. Surprisingly, H. pylori infection had no significant impact on the number of HLA-DR and CD11c MNPs or their distribution within the gastric lamina propria. However, our study demonstrated that MNPmApp is a reliable and user-friendly tool for unbiased quantitation of MNPs and their distribution at mucosal sites.

The Accessory Gene saeP of the SaeR/S Two-Component Gene Regulatory System Impacts Staphylococcus aureus Virulence During Neutrophil Interaction.

Staphylococcus aureus (S. aureus) causes a range of diseases ranging from superficial skin and soft-tissue infections to invasive and life-threatening conditions (Klevens et al., 2007; Kobayashi et al., 2015). S. aureus utilizes the Sae sensory system to adapt to neutrophil challenge. Although the roles of the SaeR response regulator and its cognate sensor kinase SaeS have been demonstrated to be critical for surviving neutrophil interaction and for causing infection, the roles for the accessory proteins SaeP and SaeQ remain incompletely defined. To characterize the functional role of these proteins during innate immune interaction, we generated isogenic deletion mutants lacking these accessory genes in USA300 (USA300ΔsaeP and USA300ΔsaeQ). S. aureus survival was increased following phagocytosis of USA300ΔsaeP compared to USA300 by neutrophils. Additionally, secreted extracellular proteins produced by USA300ΔsaeP cells caused significantly more plasma membrane damage to human neutrophils than extracellular proteins produced by USA300 cells. Deletion of saeQ resulted in a similar phenotype, but effects did not reach significance during neutrophil interaction. The enhanced cytotoxicity of USA300ΔsaeP cells toward human neutrophils correlated with an increased expression of bi-component leukocidins known to target these immune cells. A saeP and saeQ double mutant (USA300ΔsaePQ) showed a significant increase in survival following neutrophil phagocytosis that was comparable to the USA300ΔsaeP single mutant and increased the virulence of USA300 during murine bacteremia. These data provide evidence that SaeP modulates the Sae-mediated response of S. aureus against human neutrophils and suggest that saeP and saeQ together impact pathogenesis in vivo.

Quantifying the Cytotoxicity of Staphyloccus aureus Against Human Polymorphonuclear Leukocytes.

Staphylococcus aureus is capable of secreting a wide range of leukocidins that target and disrupt the membrane integrity of polymorphonuclear leukocytes (PMNs or neutrophils). This protocol describes both the purification of human PMNs and the quantification of S. aureus cytotoxicity against PMNs in three different sections. Section 1 details the isolation of PMNs and serum from human blood using density centrifugation. Section 2 tests the cytotoxicity of extracellular proteins produced by S. aureus against these purified human PMNs. Section 3 measures the cytotoxicity against human PMNs following the phagocytosis of live S. aureus. These procedures measure disruption of PMN plasma membrane integrity by S. aureus leukocidins using flow cytometry analysis of PMNs treated with propidium iodide, a DNA binding fluorophore that is cell membrane impermeable. Collectively, these methods have the advantage of rapidly testing S. aureus cytotoxicity against primary human PMNs and can be easily adapted to study other aspects of host-pathogen interactions.

CD103 (αE Integrin) Undergoes Endosomal Trafficking in Human Dendritic Cells, but Does Not Mediate Epithelial Adhesion.

Dendritic cell (DC) expression of CD103, the α subunit of αEß7 integrin, is thought to enable DC interactions with E-cadherin-expressing gastrointestinal epithelia for improved mucosal immunosurveillance. In the stomach, efficient DC surveillance of the epithelial barrier is crucial for the induction of immune responses to H. pylori, the causative agent of peptic ulcers and gastric cancer. However, gastric DCs express only low levels of surface CD103, as we previously showed. We here tested the hypothesis that intracellular pools of CD103 in human gastric DCs can be redistributed to the cell surface for engagement of epithelial cell-expressed E-cadherin to promote DC-epithelial cell adhesion. In support of our hypothesis, immunofluorescence analysis of tissue sections showed that CD103+ gastric DCs were preferentially localized within the gastric epithelial layer. Flow cytometry and imaging cytometry revealed that human gastric DCs expressed intracellular CD103, corroborating our previous findings in monocyte-derived DCs (MoDCs). Using confocal microscopy, we show that CD103 was present in endosomal compartments, where CD103 partially co-localized with clathrin, early endosome antigen-1 and Rab11, suggesting that CD103 undergoes endosomal trafficking similar to ß1 integrins. Dynamic expression of CD103 on human MoDCs was confirmed by internalization assay. To analyze whether DC-expressed CD103 promotes adhesion to E-cadherin, we performed adhesion and spreading assays on E-cadherin-coated glass slides. In MoDCs generated in the presence of retinoic acid, which express increased CD103, intracellular CD103 significantly redistributed toward the E-cadherin-coated glass surface. However, DCs spreading and adhesion did not differ between E-cadherin-coated slides and slides coated with serum alone. In adhesion assays using E-cadherin-positive HT-29 cells, DC binding was significantly improved by addition of Mn2+ and decreased in the presence of EGTA, consistent with the dependence of integrin-based interactions on divalent cations. However, retinoic acid failed to increase DC adhesion, and a CD103 neutralizing antibody was unable to inhibit DC binding to the E-cadherin positive cells. In contrast, a blocking antibody to DC-expressed E-cadherin significantly reduced DC binding to the epithelium. Overall, these data indicate that CD103 engages in DC-epithelial cell interactions upon contact with epithelial E-cadherin, but is not a major driver of DC adhesion to gastrointestinal epithelia.

Aspartic Acid Residue 51 of SaeR Is Essential for Staphylococcus aureus Virulence.

Staphylococcus aureus is a common Gram-positive bacteria that is a major cause of human morbidity and mortality. The SaeR/S two-component sensory system of S. aureus is important for virulence gene transcription and pathogenesis. However, the influence of SaeR phosphorylation on virulence gene transcription is not clear. To determine the importance of potential SaeR phosphorylation sites for S. aureus virulence, we generated genomic alanine substitutions at conserved aspartic acid residues in the receiver domain of the SaeR response regulator in clinically significant S. aureus pulsed-field gel electrophoresis (PFGE) type USA300. Transcriptional analysis demonstrated a dramatic reduction in the transcript abundance of various toxins, adhesins, and immunomodulatory proteins for SaeR with an aspartic acid to alanine substitution at residue 51. These findings corresponded to a significant decrease in cytotoxicity against human erythrocytes and polymorphonuclear leukocytes, the ability to block human myeloperoxidase activity, and pathogenesis during murine soft-tissue infection. Analysis of SaeR sequences from over 8,000 draft S. aureus genomes revealed that aspartic acid residue 51 is 100% conserved. Collectively, these results demonstrate that aspartic acid residue 51 of SaeR is essential for S. aureus virulence and underscore a conserved target for novel antimicrobial strategies that treat infection caused by this pathogen.

Clostridium difficile environmental contamination within a clinical laundry facility in the USA.

Clostridium difficile is both a hospital and community-acquired pathogen. The current study determined if C. difficile could be cultured from clinical laundry facility surfaces. A total of 240 surface samples were collected from dirty areas (n = 120), which handle soiled clinical linens, and from clean areas (n = 120), which process and fold the clean linens, within the University of Washington Consolidated Laundry facility in 2015. Sampling was done four times over the course of 1 year. The dirty area was significantly more contaminated than the clean area (21% vs 2%, P < 0.001). Clostridium difficile isolates were genetically characterized using multilocus sequence typing and PCR for the detection of genes encoding toxin A and toxin B. The MLST types 1, 2, 3, 15, 26, 34, 35, 39, 42, 43, 44, 53, 63 and 284 were identified and have previously been found in both clinical and community settings. Toxin positive isolates were identified in both the dirty (n = 16/25) and clean areas (n = 2/2). Seasonal variation was observed with 40% of the 27 isolates cultured in April 2015. The study suggests that soiled clinical linens may be a source of C. difficile surface contamination.

Transmission of MDR MRSA between primates, their environment and personnel at a United States primate centre.

OBJECTIVES: MDR MRSA isolates cultured from primates, their facility and primate personnel from the Washington National Primate Research Center were characterized to determine whether they were epidemiologically related to each other and if they represented common local human-associated MRSA strains. METHODS: Human and primate nasal and composite environmental samples were collected, enriched and selected on medium supplemented with oxacillin and polymyxin B. Isolates were biochemically verified as Staphylococcus aureus and screened for the mecA gene. Selected isolates were characterized using SCCmec typing, MLST and WGS. RESULTS: Nasal cultures were performed on 596 primates and 105 (17.6%) were MRSA positive. Two of 79 (2.5%) personnel and two of 56 (3.6%) composite primate environmental facility samples were MRSA positive. Three MRSA isolates from primates, one MRSA from personnel, two environmental MRSA and one primate MSSA were ST188 and were the same strain type by conventional typing methods. ST188 isolates were related to a 2007 ST188 human isolate from Hong Kong. Both MRSA isolates from out-of-state primates had a novel MLST type, ST3268, and an unrelated group. All isolates carried ≥1 other antibiotic resistance gene(s), including tet(38), the only tet gene identified. CONCLUSIONS: ST188 is very rare in North America and has almost exclusively been identified in people from Pan-Asia, while ST3268 is a newly reported MRSA type. The data suggest that the primate MDR MRSA was unlikely to come from primate centre employees. Captive primates are likely to be an unappreciated source of MRSA.