Faculty Bibliography

We describe a case of fetal parvovirus B19 infection resulting in preterm birth and leading to hydrops fetalis requiring multiple in utero transfusions. The infant developed chronic postnatal anemia responsive to intravenous immunoglobulin therapy. Serum viral load decreased after immunoglobulin treatment but remained detectable for over 1 year.

The contribution of specific factors to bacterial virulence is generally investigated through creation of genetic "knockouts" that are then compared to wild-type strains or complemented mutants. This paradigm is useful to understand the effect of presence vs. absence of a specific gene product but cannot account for concentration-dependent effects, such as may occur with some bacterial toxins. In order to assess threshold and dose-response effects of virulence factors, robust systems for tunable expression are required. Recent evidence suggests that the folding free energy (DeltaG) of the 5' end of mRNA transcripts can have a significant effect on translation efficiency and overall protein abundance. Here we demonstrate that rational alteration of 5' mRNA folding free energy by introduction of synonymous mutations allows for predictable changes in pneumolysin (PLY) expression by Streptococcus pneumoniae without the need for chemical inducers or heterologous promoters. We created a panel of isogenic S. pneumoniae strains, differing only in synonymous (silent) mutations at the 5' end of the PLY mRNA that are predicted to alter DeltaG. Such manipulation allows rheostat-like control of PLY production and alters the cytotoxicity of whole S. pneumoniae on primary and immortalized human cells. These studies provide proof-of-principle for further investigation of mRNA DeltaG manipulation as a tool in studies of bacterial pathogenesis.

Streptococcus agalactiae (group B Streptococcus [GBS]) is a human pathogen with a propensity to cause neonatal infections. We report the complete genome sequence of GBS strain CNCTC 10/84, a hypervirulent clinical isolate frequently used to study GBS pathogenesis. Comparative analysis of this sequence may shed light on novel pathogenic mechanisms.

Background Bacterial vaginosis (BV) is the most common reproductive tract infection (RTI) and is a significant risk factor for preterm birth. Microbicides could be an option for the prevention and treatment of BV in pregnancy, and understanding use of the product will be crucial. The present study explored attitudes of women in the third trimester of pregnancy regarding topical microbicide use for the prevention and treatment of BV. METHODS: Twenty-six women in their third trimester were interviewed regarding their knowledge and beliefs about RTIs during pregnancy and attitudes concerning the use of topical microbicides for prevention and treatment of BV. RESULTS: Participants had a mean age of 24.9 years, were largely under-represented minorities and the majority had had past pregnancies. Participants had knowledge and experience with RTIs but not BV. They were open to the use of microbicides for prevention or treatment of BV, but believed that women requiring treatment would be more motivated. Rationales for acceptability were most commonly related to the baby's health. Practical issues that may interfere with use were often, but not always, related to pregnancy. There was a range of attitudes about partner involvement in decision-making and the practicalities of product use. CONCLUSION: Pregnant women are knowledgeable about RTIs but not necessarily BV. The women in this study found microbicide use acceptable, particularly for treatment. To improve acceptability and use, education would be needed about BV and possible complications, how to overcome practical problems and the value of involving partners in the decision.

A subgroup of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins (PFTs) has an unusually narrow host range due to a requirement for binding to human CD59 (hCD59), a glycosylphosphatidylinositol (GPI)-linked complement regulatory molecule. hCD59-specific CDCs are produced by several organisms that inhabit human mucosal surfaces and can act as pathogens, including Gardnerella vaginalis and Streptococcus intermedius. The consequences and potential selective advantages of such PFT host limitation have remained unknown. Here, we demonstrate that, in addition to species restriction, PFT ligation of hCD59 triggers a previously unrecognized pathway for programmed necrosis in primary erythrocytes (red blood cells [RBCs]) from humans and transgenic mice expressing hCD59. Because they lack nuclei and mitochondria, RBCs have typically been thought to possess limited capacity to undergo programmed cell death. RBC programmed necrosis shares key molecular factors with nucleated cell necroptosis, including dependence on Fas/FasL signaling and RIP1 phosphorylation, necrosome assembly, and restriction by caspase-8. Death due to programmed necrosis in RBCs is executed by acid sphingomyelinase-dependent ceramide formation, NADPH oxidase- and iron-dependent reactive oxygen species formation, and glycolytic formation of advanced glycation end products. Bacterial PFTs that are hCD59 independent do not induce RBC programmed necrosis. RBC programmed necrosis is biochemically distinct from eryptosis, the only other known programmed cell death pathway in mature RBCs. Importantly, RBC programmed necrosis enhances the growth of PFT-producing pathogens during exposure to primary RBCs, consistent with a role for such signaling in microbial growth and pathogenesis. IMPORTANCE: In this work, we provide the first description of a new form of programmed cell death in erythrocytes (RBCs) that occurs as a consequence of cellular attack by human-specific bacterial toxins. By defining a new RBC death pathway that shares important components with necroptosis, a programmed necrosis module that occurs in nucleated cells, these findings expand our understanding of RBC biology and RBC-pathogen interactions. In addition, our work provides a link between cholesterol-dependent cytolysin (CDC) host restriction and promotion of bacterial growth in the presence of RBCs, which may provide a selective advantage to human-associated bacterial strains that elaborate such toxins and a potential explanation for the narrowing of host range observed in this toxin family.

BACKGROUND: Maternal vaginal colonization with Streptococcus agalactiae (Group B Streptococcus [GBS]) is a precursor to chorioamnionitis, fetal infection, and neonatal sepsis, but the understanding of specific factors in the pathogenesis of ascending infection remains limited. METHODS: We used a new murine model to evaluate the contribution of the pore-forming GBS beta-hemolysin/cytolysin (betaH/C) to vaginal colonization, ascension, and fetal infection. RESULTS: Competition assays demonstrated a marked advantage to betaH/C-expressing GBS during colonization. Intrauterine fetal demise and/or preterm birth were observed in 54% of pregnant mice colonized with wild-type (WT) GBS and 0% of those colonized with the toxin-deficient cylE knockout strain, despite efficient colonization and ascension by both strains. Robust placental inflammation, disruption of maternal-fetal barriers, and fetal infection were more frequent in animals colonized with WT bacteria. Histopathologic examination revealed bacterial tropism for fetal lung and liver. CONCLUSIONS: Preterm birth and fetal demise are likely the direct result of toxin-induced damage and inflammation rather than differences in efficiency of ascension into the upper genital tract. These data demonstrate a distinct contribution of betaH/C to GBS chorioamnionitis and subsequent fetal infection in vivo and showcase a model for this most proximal step in GBS pathogenesis.

alpha-Intercalated cells (A-ICs) within the collecting duct of the kidney are critical for acid-base homeostasis. Here, we have shown that A-ICs also serve as both sentinels and effectors in the defense against urinary infections. In a murine urinary tract infection model, A-ICs bound uropathogenic E. coli and responded by acidifying the urine and secreting the bacteriostatic protein lipocalin 2 (LCN2; also known as NGAL). A-IC-dependent LCN2 secretion required TLR4, as mice expressing an LPS-insensitive form of TLR4 expressed reduced levels of LCN2. The presence of LCN2 in urine was both necessary and sufficient to control the urinary tract infection through iron sequestration, even in the harsh condition of urine acidification. In mice lacking A-ICs, both urinary LCN2 and urinary acidification were reduced, and consequently bacterial clearance was limited. Together these results indicate that A-ICs, which are known to regulate acid-base metabolism, are also critical for urinary defense against pathogenic bacteria. They respond to both cystitis and pyelonephritis by delivering bacteriostatic chemical agents to the lower urinary system.

Staphylococcus aureus 502A was a strain used in bacterial interference programs during the 1960s and early 1970s. Infants were deliberately colonized with 502A with the goal of preventing colonization with more invasive strains. We present the completed genome sequence of this organism.

Streptococcus agalactiae (group B Streptococcus, GBS) usually colonizes the gastrointestinal and lower genital tracts of asymptomatic hosts, yet the incidence of invasive disease is on the rise . We describe a case of an 18 year old woman, recently diagnosed with lupus, who reported a spontaneous abortion six weeks prior to her hospitalization. She presented with fever, altered mental status, and meningeal signs, paired with a positive blood culture for GBS. Magnetic resonance imaging of her brain demonstrated an extra-axial fluid collection, and she was diagnosed with meningitis. She received prolonged intravenous antibiotic therapy and aggressive treatment for lupus, leading to clinical recovery. This case illustrates the importance of recognizing GBS as a potential pathogen in all patients presenting with CNS infection .