Summary Streptococcus agalactiae (Group B Streptococcus, GBS) is a frequent commensal organism of the vaginal tract of healthy women. However, GBS can transition to a pathogen in susceptible hosts, but host and microbial factors that contribute to this conversion are not well understood. GBS CovR/S (CsrR/S) is a two component regulatory system that regulates key virulence elements including adherence and toxin production. We performed global transcription profiling of human vaginal epithelial cells exposed to WT, CovR deficient, and toxin deficient strains, and observed that insufficient regulation by CovR and subsequent increased toxin production results in a drastic increase in host inflammatory responses, particularly in cytokine signaling pathways promoted by IL-8 and CXCL2. Additionally, we observed that CovR regulation impacts epithelial cell attachment and intracellular invasion. In our mouse model of GBS vaginal colonization, we further demonstrated that CovR regulation promotes vaginal persistence, as infection with a CovR deficient strain resulted in a heightened host immune response as measured by cytokine production and neutrophil activation. Using CXCr2 KO mice, we determined that this immune alteration occurs, at least in part, via signaling through the CXCL2 receptor. Taken together, we conclude that CovR is an important regulator of GBS vaginal colonization and loss of this regulatory function may contribute to the inflammatory havoc seen during the course of infection.

Streptococcus agalactiae (group B streptococcus [GBS]) is a Gram-positive bacterium found in the female rectovaginal tract and is capable of producing severe disease in susceptible hosts, including newborns and pregnant women. The vaginal tract is considered a major reservoir for GBS, and maternal vaginal colonization poses a significant risk to the newborn; however, little is known about the specific bacterial factors that promote GBS colonization and persistence in the female reproductive tract. We have developed in vitro models of GBS interaction with the human female cervicovaginal tract using human vaginal and cervical epithelial cell lines. Analysis of isogenic mutant GBS strains deficient in cell surface organelles such as pili and serine-rich repeat (Srr) proteins shows that these factors contribute to host cell attachment. As Srr proteins are heavily glycosylated, we confirmed that carbohydrate moieties contribute to the effective interaction of Srr-1 with vaginal epithelial cells. Antibody inhibition assays identified keratin 4 as a possible host receptor for Srr-1. Our findings were further substantiated in an in vivo mouse model of GBS vaginal colonization, where mice inoculated with an Srr-1-deficient mutant exhibited decreased GBS vaginal persistence compared to those inoculated with the wild-type (WT) parental strain. Furthermore, competition experiments in mice showed that WT GBS exhibited a significant survival advantage over the ⌬pilA or ⌬srr-1 mutant in the vaginal tract. Our results suggest that these GBS surface proteins contribute to vaginal colonization and may offer new insights into the mechanisms of vaginal niche establishment.Group B streptococcus (GBS) is the leading cause of neonatal meningitis and sepsis in the developed world and also causes serious invasive infections in certain adult populations (54). GBS can be isolated from the rectovaginal tracts of up to 30% of women (16,38), and it can be transmitted to infants during birth through the aspiration of vaginal fluids or cross the placental barrier in utero (7,18). GBS neonatal infection is divided into two categories, early-onset (Ͻ7 days old) and late-onset (7 to 90 days old) disease. Due to the serious nature of GBS infection, pregnant women in the United States are routinely screened for GBS vaginal colonization late in the third trimester of pregnancy; a positive test results in the administration of antibiotics during birth to reduce the risk of GBS transfer to the newborn. Despite this intervention, the incidence of early-onset GBS infection in the United States remains at 1 in 3,000 live births, corresponding to approximately 1,200 infected infants per year (54). There is also evidence that infection rates are much higher among some ethnic groups and in infants delivered at Ͻ37 weeks of gestation (42,43,54,62). Additionally, antibiotic prophylaxis does not prevent late-onset disease.Most women are intermittently asymptomatically colonized by GBS in the genitourinary tract (19); however, colonization poses a significant r...

Membrane-bound organelles serve as platforms for the assembly of multi-protein complexes that function as hubs of signal transduction in eukaryotic cells. Microbial pathogens have evolved virulence factors that reprogram these host signaling responses, but the underlying molecular mechanisms are poorly understood. Here we test the ability of ~200 type III and type IV effector proteins from six Gram-negative bacterial species to interact with the eukaryotic plasma membrane and intracellular organelles. We show that over 30% of the effectors localize to yeast and mammalian cell membranes, including a subset of previously uncharacterized Legionella effectors that appear to be able to regulate yeast vacuolar fusion. A combined genetic, cellular, and biochemical approach supports that some of the tested bacterial effectors can bind to membrane phospholipids and may regulate membrane trafficking. Finally, we show that the type III effector IpgB1 from Shigella flexneri may bind to acidic phospholipids and regulate actin filament dynamics.

The type I interferon (IFN) activated transcriptional response is a critical antiviral defense mechanism, yet its role in bacterial pathogenesis remains less well characterized. Using an intracellular pathogen Listeria monocytogenes (Lm) as a model bacterial pathogen, we sought to identify the roles of individual interferon-stimulated genes (ISGs) in context of bacterial infection. Previously, IFN has been implicated in both restricting and promoting Lm growth and immune stimulatory functions in vivo. Here we adapted a gain-of-function flow cytometry based approach to screen a library of more than 350 human ISGs for inhibitors and enhancers of Lm infection. We identify 6 genes, including UNC93B1, MYD88, AQP9, and TRIM14 that potently inhibit Lm infection. These inhibitors act through both transcription-mediated (MYD88) and non-transcriptional mechanisms (TRIM14). Further, we identify and characterize the human high affinity immunoglobulin receptor FcγRIa as an enhancer of Lm internalization. Our results reveal that FcγRIa promotes Lm uptake in the absence of known host Lm internalization receptors (E-cadherin and c-Met) as well as bacterial surface internalins (InlA and InlB). Additionally, FcγRIa-mediated uptake occurs independently of Lm opsonization or canonical FcγRIa signaling. Finally, we established the contribution of FcγRIa to Lm infection in phagocytic cells, thus potentially linking the IFN response to a novel bacterial uptake pathway. Together, these studies provide an experimental and conceptual basis for deciphering the role of IFN in bacterial defense and virulence at single-gene resolution.

Bacterial pathogens encode a wide variety of effectors and toxins that hijack host cell structure and function. Of particular importance are virulence factors that target actin cytoskeleton dynamics critical for cell shape, stability, motility, phagocytosis, and division. In addition, many bacteria target organelles of the general secretory pathway (e.g., the endoplasmic reticulum and the Golgi complex) and recycling pathways (e.g., the endolysosomal system) to establish and maintain an intracellular replicative niche. Recent research on the biochemistry and structural biology of bacterial effector proteins and toxins has begun to shed light on the molecular underpinnings of these host-pathogen interactions. This exciting work is revealing how pathogens gain control of the complex and dynamic host cellular environments, which impacts our understanding of microbial infectious disease, immunology, and human cell biology.