Campylobacter jejuni, a gram-negative motile bacterium, secretes a set of proteins termed the Campylobacter invasion antigens (Cia proteins). The purpose of this study was to determine whether the flagellar apparatus serves as the export apparatus for the Cia proteins. Mutations were generated in five genes encoding three structural components of the flagella, the flagellar basal body (flgB and flgC), hook (flgE2), and filament (flaA and flaB) genes, as well as in genes whose products are essential for flagellar protein export (flhB and fliI). While mutations that affected filament assembly were found to be nonmotile (Mot ؊ ) and did not secrete Cia proteins (S ؊ ), a flaA (flaB ؉ ) filament mutant was found to be nonmotile but Cia protein secretion competent (Mot ؊ , S ؉ ). Complementation of a flaA flaB double mutant with a shuttle plasmid harboring either the flaA or flaB gene restored Cia protein secretion, suggesting that Cia export requires at least one of the two filament proteins. Infection of INT 407 human intestinal cells with the C. jejuni mutants revealed that maximal invasion of the epithelial cells required motile bacteria that are secretion competent. Collectively, these data suggest that the C. jejuni Cia proteins are secreted from the flagellar export apparatus.Campylobacter jejuni, a gram-negative motile bacterium, is a frequent cause of human gastrointestinal infections (39). The spectrum of disease observed in C. jejuni-infected individuals ranges from asymptomatic to severe enteritis characterized by fever, severe abdominal cramping, and diarrhea with blood and mucus (2, 4). By analogy with other more extensively characterized bacterial pathogens, the mechanism of C. jejunimediated enteritis is proposed to be multifactorial. Previous work has indicated that motility as well as the presence of the flagellum contributes to the ability of C. jejuni to colonize the intestinal tract of animals (33,36,42).The flagellum of C. jejuni is composed of a basal body, hook, and filament. The flagellar filament is comprised of two proteins, FlaA and FlaB, although it appears that FlaA is the preferred subunit (3). While the C. jejuni FlaA and FlaB flagellin proteins are transcribed concomitantly (16), the flaA gene is regulated by 28 and the flaB gene is regulated by 54 (3, 16). Hendrixson et al. (16) noted that a C. jejuni isolate deficient in 28 , which is encoded by the fliA gene, is able to assemble a truncated filament composed exclusively of the flagellin protein FlaB. This result indicates that the regulation of flagellar gene expression within C. jejuni differs from the regulation in more intensely studied systems such as that of Salmonella enterica. Unlike flagellar gene expression in C. jejuni, flagellar gene expression in S. enterica is initiated by a master regulator, while late gene expression and motility require 28 (1). Previous work in our laboratory has demonstrated that C. jejuni synthesizes a set of proteins during coculture with epithelial cells, a subset of which are secreted. The sec...

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Historically, non-combat injuries and illnesses have had a significant impact on military missions. We conducted an anonymous cross-sectional survey to assess the prevalence and impact of common ailments among U.S. military personnel deployed to Iraq or Afghanistan during 2003-2004. Among 15,459 persons surveyed, diarrhea (76.8% in Iraq and 54.4% in Afghanistan), respiratory illness (69.1%), non-combat injuries (34.7%), and leishmaniasis (2.1%) were commonly reported. For all causes, 25.2% reported that they required intravenous fluids, 10.4% required hospitalization, and 5.2% required medical evacuation. Among ground units, 12.7% reported that they missed a patrol because of illness, and among air units, 11.7% were grounded because of illness. The incidence of diarrhea and respiratory infections doubled from the pre-combat to combat phases, and the perceived adverse impact of these illnesses on the unit increased significantly during the combat phase. Despite technologic advances in warfare and preventive medicine, illness and non-combat injuries have been common during operations in Iraq and Afghanistan, resulting in frequent transient decreases in operational efficiency.

BackgroundEffective influenza surveillance requires new methods capable of rapid and inexpensive genomic analysis of evolving viral species for pandemic preparedness, to understand the evolution of circulating viral species, and for vaccine strain selection. We have developed one such approach based on previously described broad-range reverse transcription PCR/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology.Methods and Principal FindingsAnalysis of base compositions of RT-PCR amplicons from influenza core gene segments (PB1, PB2, PA, M, NS, NP) are used to provide sub-species identification and infer influenza virus H and N subtypes. Using this approach, we detected and correctly identified 92 mammalian and avian influenza isolates, representing 30 different H and N types, including 29 avian H5N1 isolates. Further, direct analysis of 656 human clinical respiratory specimens collected over a seven-year period (1999–2006) showed correct identification of the viral species and subtypes with >97% sensitivity and specificity. Base composition derived clusters inferred from this analysis showed 100% concordance to previously established clades. Ongoing surveillance of samples from the recent influenza virus seasons (2005–2006) showed evidence for emergence and establishment of new genotypes of circulating H3N2 strains worldwide. Mixed viral quasispecies were found in approximately 1% of these recent samples providing a view into viral evolution.Conclusion/SignificanceThus, rapid RT-PCR/ESI-MS analysis can be used to simultaneously identify all species of influenza viruses with clade-level resolution, identify mixed viral populations and monitor global spread and emergence of novel viral genotypes. This high-throughput method promises to become an integral component of influenza surveillance.

Previous studies have indicated that the ability to bind to fibronectin is a key feature in successful cell invasion by Campylobacter jejuni. Given the spatial distribution of fibronectin and the architecture of the epithelium, this suggests the possibility that C. jejuni cell invasion might preferentially occur at the basolateral cell surface. To test this hypothesis, we examined the interaction of C. jejuni with T84 human colonic cells. When grown under the appropriate conditions, T84 cells form a polarized cell monolayer. C. jejuni translocation of a T84 cell monolayer appeared to occur via a paracellular (extracellular) route as opposed to a transcellular (intracellular) route based on the finding that a C. jejuni noninvasive mutant translocated as efficiently as its isogenic parent. Additional studies revealed that two distinct C. jejuni wild-type isolates could compete with one another for host cell receptors, whereas a C. jejuni fibronectin-binding-deficient mutant could not compete with a wild-type isolate for host cell receptors. Further, C. jejuni adherence and internalization were significantly inhibited by antifibronectin antibodies but only when cells were first treated with EGTA to expose basolateral cell surfaces. Together, these results support the theory that C. jejuni invasion occurs preferentially at the basolateral surface of eukaryotic cells.