Faculty Bibliography

Pore-forming toxins (PFTs) form a large group of bacterial virulence factors that play an important role in various infectious diseases. These include infections with problematic pathogens such as Streptococcus pneumoniae, Staphylococcus aureus, group A and B streptococci, Escherichia coli and Mycobacterium tuberculosis. PFTs perforate host cell membranes, which contributes to the establishment or exacerbation of an infection mainly in two ways: first, by disrupting the host immune response, and second, by helping bacteria to cross epithelial and endothelial barriers, thus allowing them to spread to other parts of the host. Although perforation of the plasma membrane can lead to host cell death, cells possess molecular defence mechanisms and under certain conditions can successfully defend themselves against PFTs. PFTs, as well as the immune response against PFTs, form a potential target for novel prophylactics and therapeutics against bacterial infectious disease, including against antibiotic-resistant strains.

OBJECTIVE: Female genital tract secretions inhibit E. coli ex vivo and the activity may prevent colonization and provide a biomarker of a healthy microbiome. We hypothesized that high E. coli inhibitory activity would be associated with a Lactobacillus crispatus and/or jensenii dominant microbiome and differ from that of women with low inhibitory activity. STUDY DESIGN: Vaginal swab cell pellets from 20 samples previously obtained in a cross-sectional study of near-term pregnant and non-pregnant healthy women were selected based on having high (>90% inhibition) or low (<20% inhibition) anti-E. coli activity. The V6 region of the 16S ribosomal RNA gene was amplified and sequenced using the Illumina HiSeq 2000 platform. Filtered culture supernatants from Lactobacillus crispatus, Lactobacillus iners, and Gardnerella vaginalis were also assayed for E. coli inhibitory activity. RESULTS: Sixteen samples (10 with high and 6 with low activity) yielded evaluable microbiome data. There was no difference in the predominant microbiome species in pregnant compared to non-pregnant women (n = 8 each). However, there were significant differences between women with high compared to low E. coli inhibitory activity. High activity was associated with a predominance of L. crispatus (p<0.007) and culture supernatants from L. crispatus exhibited greater E. coli inhibitory activity compared to supernatants obtained from L. iners or G. vaginalis. Notably, the E. coli inhibitory activity varied among different strains of L. crispatus. CONCLUSION: Microbiome communities with abundant L. crispatus likely contribute to the E. coli inhibitory activity of vaginal secretions and efforts to promote this environment may prevent E. coli colonization and related sequelae including preterm birth.

The arginine catabolic mobile element (ACME) is the largest genomic region distinguishing epidemic USA300 strains of methicillin-resistant Staphylococcus aureus (MRSA) from other S. aureus strains. However, the functional relevance of ACME to infection and disease has remained unclear. Using phylogenetic analysis, we have shown that the modular segments of ACME were assembled into a single genetic locus in Staphylococcus epidermidis and then horizontally transferred to the common ancestor of USA300 strains in an extremely recent event. Acquisition of one ACME gene, speG, allowed USA300 strains to withstand levels of polyamines (e.g., spermidine) produced in skin that are toxic to other closely related S. aureus strains. speG-mediated polyamine tolerance also enhanced biofilm formation, adherence to fibrinogen/fibronectin, and resistance to antibiotic and keratinocyte-mediated killing. We suggest that these properties gave USA300 a major selective advantage during skin infection and colonization, contributing to the extraordinary evolutionary success of this clone. IMPORTANCE: Over the past 15 years, methicillin-resistant Staphylococcus aureus (MRSA) has become a major public health problem. It is likely that adaptations in specific MRSA lineages (e.g., USA300) drove the spread of MRSA across the United States and allowed it to replace other, less-virulent S. aureus strains. We suggest that one major factor in the evolutionary success of MRSA may have been the acquisition of a gene (speG) that allows S. aureus to evade the toxicity of polyamines (e.g., spermidine and spermine) that are produced in human skin. Polyamine tolerance likely gave MRSA multiple fitness advantages, including the formation of more-robust biofilms, increased adherence to host tissues, and resistance to antibiotics and killing by human skin cells.

Gardnerella vaginalis, the bacterial species most frequently isolated from women with bacterial vaginosis (BV), produces a cholesterol-dependent cytolysin (CDC), vaginolysin (VLY). At sublytic concentrations, CDCs may initiate complex signaling cascades crucial to target cell survival. Using live-cell imaging, we observed the rapid formation of large membrane blebs in human vaginal and cervical epithelial cells (VK2 and HeLa cells) exposed to recombinant VLY toxin and to cell-free supernatants from growing liquid cultures of G. vaginalis. Binding of VLY to its human-specific receptor (hCD59) is required for bleb formation, as antibody inhibition of either toxin or hCD59 abrogates this response, and transfection of nonhuman cells (CHO-K1) with hCD59 renders them susceptible to toxin-induced membrane blebbing. Disruption of the pore formation process (by exposure to pore-deficient toxoids or pretreatment of cells with methyl-beta-cyclodextrin) or osmotic protection of target cells inhibits VLY-induced membrane blebbing. These results indicate that the formation of functional pores drives the observed ultrastructural rearrangements. Rapid bleb formation may represent a conserved response of epithelial cells to sublytic quantities of pore-forming toxins, and VLY-induced epithelial cell membrane blebbing in the vaginal mucosa may play a role in the pathogenesis of BV.

Pore-forming toxins (PFTs) are the most common bacterial cytotoxic proteins and are required for virulence in a large number of important pathogens, including Streptococcus pneumoniae, group A and B streptococci, Staphylococcus aureus, Escherichia coli, and Mycobacterium tuberculosis. PFTs generally disrupt host cell membranes, but they can have additional effects independent of pore formation. Substantial effort has been devoted to understanding the molecular mechanisms underlying the functions of certain model PFTs. Likewise, specific host pathways mediating survival and immune responses in the face of toxin-mediated cellular damage have been delineated. However, less is known about the overall functions of PFTs during infection in vivo. This review focuses on common themes in the area of PFT biology, with an emphasis on studies addressing the roles of PFTs in in vivo and ex vivo models of colonization or infection. Common functions of PFTs include disruption of epithelial barrier function and evasion of host immune responses, which contribute to bacterial growth and spreading. The widespread nature of PFTs make this group of toxins an attractive target for the development of new virulence-targeted therapies that may have broad activity against human pathogens.

Bacterial vaginosis is a highly prevalent and poorly understood polymicrobial disorder of the vaginal microbiota, with significant adverse sequelae. Gardnerella vaginalis predominates in bacterial vaginosis. Biofilms of G. vaginalis are present in human infections and are implicated in persistent disease, treatment failure, and transmission. Here we demonstrate that G. vaginalis biofilms contain extracellular DNA, which is essential to their structural integrity. Enzymatic disruption of this DNA specifically inhibits biofilms, acting on both newly forming and established biofilms. DNase liberates bacteria from the biofilm to supernatant fractions and potentiates the activity of metronidazole, an antimicrobial agent used in the treatment of bacterial vaginosis. Using a new murine vaginal colonization model for G. vaginalis, we demonstrate >10-fold inhibition of G. vaginalis colonization by DNase. We conclude that DNase merits investigation as a potential nonantibiotic adjunct to existing bacterial vaginosis therapies in order to decrease the risk of chronic infection, recurrence, and associated morbidities.

Bordetella holmesii is an emerging opportunistic pathogen that causes respiratory disease in healthy individuals and invasive infections among patients lacking splenic function. We used 16S rRNA gene analysis to confirm B. holmesii as the cause of bacteremia in a child with sickle cell disease. Semiconductor-based draft genome sequencing provided insight into B. holmesii phylogeny and potential virulence mechanisms and also identified a toluene-4-monoxygenase locus unique among bordetellae.

Klebsiella pneumoniae K1 is a major agent of hepatic abscess with metastatic disease in East Asia, with sporadic reports originating elsewhere. We report a case of abscess complicated by septic endophthalmitis caused by a wzyAKpK1-positive Klebsiella strain in a U.S. resident, raising concern for global emergence.

Streptococcus intermedius is a human pathogen with a propensity for abscess formation. We report a high-quality draft genome sequence of S. intermedius strain BA1, an isolate from a human epidural abscess. This sequence provides insight into the biology of S. intermedius and will aid investigations of pathogenicity.

Group B Streptococcus (GBS; Streptococcus agalactiae) is a major human pathogen that disproportionately affects neonates and women in the peripartum period and is an emerging cause of infection in older adults. The primary toxin of GBS, beta-hemolysin/cytolysin (betaH/C), has a well-defined role in the pathogenesis of invasive disease, but its role in urinary tract infection (UTI) is unknown. Using both in vitro and in vivo models, we analyzed the importance of betaH/C in GBS uropathogenesis. There were no significant differences in bacterial density from the bladders or kidneys from mice infected with wild-type or isogenic betaH/C-deficient GBS, and competitive indices from co-infection experiments were near 1. Thus, betaH/C is dispensable for the establishment of GBS-UTI. However, betaH/C-sufficient GBS induced a more robust proinflammatory cytokine response in cultured bladder epithelial cells and in the urinary tracts of infected mice. Given the near ubiquity of betaH/C-expressing strains in epidemiologic studies and the importance of local inflammation in dictating outcomes and sequelae of UTI, we hypothesize that betaH/C-driven inflammatory signaling may be important in the clinical course of GBS-UTI.