Person:

Ryan, Edward

Background: Salmonella enterica serotype Paratyphi A is a human-restricted cause of paratyphoid fever, accounting for up to a fifth of all cases of enteric fever in Asia. Methodology/Principal Findings: In this work, we applied an RNA analysis method, Selective Capture of Transcribed Sequences (SCOTS), and cDNA hybridization-microarray technology to identify S. Paratyphi A transcripts expressed by bacteria in the blood of three patients in Bangladesh. In total, we detected 1,798 S. Paratyphi A mRNAs expressed in the blood of infected humans (43.9% of the ORFeome). Of these, we identified 868 in at least two patients, and 315 in all three patients. S. Paratyphi A transcripts identified in at least two patients encode proteins involved in energy metabolism, nutrient and iron acquisition, vitamin biosynthesis, stress responses, oxidative stress resistance, and pathogenesis. A number of detected transcripts are expressed from PhoP and SlyA-regulated genes associated with intra-macrophage survival, genes contained within Salmonella Pathogenicity Islands (SPIs) 1–4, 6, 10, 13, and 16, as well as RpoS-regulated genes. The largest category of identified transcripts is that of encoding proteins with unknown function. When comparing levels of bacterial mRNA using in vivo samples collected from infected patients to samples from in vitro grown organisms, we found significant differences for 347, 391, and 456 S. Paratyphi A transcripts in each of three individual patients (approximately 9.7% of the ORFeome). Of these, expression of 194 transcripts (4.7% of ORFs) was concordant in two or more patients, and 41 in all patients. Genes encoding these transcripts are contained within SPI-1, 3, 6 and 10, PhoP-regulated genes, involved in energy metabolism, nutrient acquisition, drug resistance, or uncharacterized genes. Using quantitative RT-PCR, we confirmed increased gene expression in vivo for a subset of these genes. Conclusion/Significance: To our knowledge, we describe the first microarray-based transcriptional analysis of a pathogen in the blood of naturally infected humans.

Background: As a result of poor economic opportunities and an increasing shortage of affordable housing, much of the spatial growth in many of the world's fastest-growing cities is a result of the expansion of informal settlements where residents live without security of tenure and with limited access to basic infrastructure. Although inadequate water and sanitation facilities, crowding and other poor living conditions can have a significant impact on the spread of infectious diseases, analyses relating these diseases to ongoing global urbanization, especially at the neighborhood and household level in informal settlements, have been infrequent. To begin to address this deficiency, we analyzed urban environmental data and the burden of cholera in Dar es Salaam, Tanzania. Methodology/Principal Findings: Cholera incidence was examined in relation to the percentage of a ward's residents who were informal, the percentage of a ward's informal residents without an improved water source, the percentage of a ward's informal residents without improved sanitation, distance to the nearest cholera treatment facility, population density, median asset index score in informal areas, and presence or absence of major roads. We found that cholera incidence was most closely associated with informal housing, population density, and the income level of informal residents. Using data available in this study, our model would suggest nearly a one percent increase in cholera incidence for every percentage point increase in informal residents, approximately a two percent increase in cholera incidence for every increase in population density of 1000 people per km2 in Dar es Salaam in 2006, and close to a fifty percent decrease in cholera incidence in wards where informal residents had minimally improved income levels, as measured by ownership of a radio or CD player on average, in comparison to wards where informal residents did not own any items about which they were asked. In this study, the range of access to improved sanitation and improved water sources was quite narrow at the ward level, limiting our ability to discern relationships between these variables and cholera incidence. Analysis at the individual household level for these variables would be of interest. Conclusions/Significance: Our results suggest that ongoing global urbanization coupled with urban poverty will be associated with increased risks for certain infectious diseases, such as cholera, underscoring the need for improved infrastructure and planning as the world's urban population continues to expand.

Background: Despite recent progress in understanding the molecular basis of Vibrio cholerae pathogenesis, there is relatively little knowledge of the factors that determine the variability in human susceptibility to V. cholerae infection. Methods and Findings: We performed an observational study of a cohort of household contacts of cholera patients in Bangladesh, and compared the baseline characteristics of household members who went on to develop culture-positive V. cholerae infection with individuals who did not develop infection. Although the vibriocidal antibody is the only previously described immunologic marker associated with protection from V. cholerae infection, we found that levels of serum IgA specific to three V. cholerae antigens—the B subunit of cholera toxin, lipopolysaccharide, and TcpA, the major component of the toxin–co-regulated pilus—also predicted protection in household contacts of patients infected with V. cholerae O1, the current predominant cause of cholera. Circulating IgA antibodies to TcpA were also associated with protection from V. cholerae O139 infection. In contrast, there was no association between serum IgG antibodies specific to these three antigens and protection from infection with either serogroup. We also found evidence that host genetic characteristics and serum retinol levels modify susceptibility to V. cholerae infection. Conclusions: Our observation that levels of serum IgA (but not serum IgG) directed at certain V. cholerae antigens are associated with protection from infection underscores the need to better understand anti–V. cholerae immunity at the mucosal surface. Furthermore, our data suggest that susceptibility to V. cholerae infection is determined by a combination of immunologic, nutritional, and genetic characteristics; additional factors that influence susceptibility to cholera remain unidentified.

Background: S. Typhi, a human-restricted Salmonella enterica serovar, causes a systemic intracellular infection in humans (typhoid fever). In comparison, S. Typhimurium causes gastroenteritis in humans, but causes a systemic typhoidal illness in mice. The PhoP regulon is a well studied two component (PhoP/Q) coordinately regulated network of genes whose expression is required for intracellular survival of S. enterica. Methodology/Principal Findings: Using high performance liquid chromatography mass spectrometry (HPLC-MS/MS), we examined the protein expression profiles of three sequenced S. enterica strains: S. Typhimurium LT2, S. Typhi CT18, and S. Typhi Ty2 in PhoP-inducing and non-inducing conditions in vitro and compared these results to profiles of (phoP^−/Q^−) mutants derived from S. Typhimurium LT2 and S. Typhi Ty2. Our analysis identified 53 proteins in S. Typhimurium LT2 and 56 proteins in S. Typhi that were regulated in a PhoP-dependent manner. As expected, many proteins identified in S. Typhi demonstrated concordant differential expression with a homologous protein in S. Typhimurium. However, three proteins (HlyE, STY1499, and CdtB) had no homolog in S. Typhimurium. HlyE is a pore-forming toxin. STY1499 encodes a stably expressed protein of unknown function transcribed in the same operon as HlyE. CdtB is a cytolethal distending toxin associated with DNA damage, cell cycle arrest, and cellular distension. Gene expression studies confirmed up-regulation of mRNA of HlyE, STY1499, and CdtB in S. Typhi in PhoP-inducing conditions. Conclusions/Significance: This study is the first protein expression study of the PhoP virulence associated regulon using strains of Salmonella mutant in PhoP, has identified three Typhi-unique proteins (CdtB, HlyE and STY1499) that are not present in the genome of the wide host-range Typhimurium, and includes the first protein expression profiling of a live attenuated bacterial vaccine studied in humans (Ty800).

Background: Infection with intestinal helminths is common and may contribute to the decreased efficacy of Vibrio cholerae vaccines in endemic compared to non-endemic areas. However, the immunomodulatory effects of concomitant intestinal parasitic infection in cholera patients have not been systematically evaluated. Methods: We evaluated V. cholerae-specific immune responses in a cohort of patients with severe cholera. 361 patients completed 21 days of observation and 53 (15%) had evidence of a concomitant intestinal parasitic infection based on direct microscopy. Although there were no significant differences in the vibriocidal or lipopolysaccharide (LPS)-specific immune responses to V. cholerae, helminth-infected cholera patients had decreased fecal and serum IgA immune responses to the B subunit of cholera toxin (CTB) as well as a more modest decrease in serum IgG response to CTB. These findings remained significant for all classes of helminth infection and when controlling for potential confounding variables such as age and nutritional status. Although we hypothesized the differential effect on CTB and LPS immune responses was due to T-cell-dependent immunomodulatory effects of helminth infection, we did not find additional evidence to support a classic Th1 or Th2 polarization of the immune response to V. cholerae infection related to parasite infection. Conclusions/Significance: The finding that helminth infection has a profound association with the mucosal humoral immune response to V. cholerae has implications for the development of protective immunity in cholera-endemic areas and provides an additional basis for deworming programs in cholera-endemic areas. Additional studies, including further characterization of the role of T cells in the immune response to human V. cholerae infection and the development of an animal model of co-infection, may provide additional insight into the mechanisms underlying these findings.

Immigrants returning home to visit friends and relatives (VFR travelers) are at higher risk of travel-associated illness than other international travelers. We evaluated 3,707 VFR and 17,507 non-VFR travelers seen for pre-travel consultation in Global TravEpiNet during 2009–2011; all were traveling to resource-poor destinations. VFR travelers more commonly visited urban destinations than non-VFR travelers (42% versus 30%, P < 0.0001); 54% of VFR travelers were female, and 18% of VFR travelers were under 6 years old. VFR travelers sought health advice closer to their departure than non-VFR travelers (median days before departure was 17 versus 26, P < 0.0001). In multivariable analysis, being a VFR traveler was an independent predictor of declining a recommended vaccine. Missed opportunities for vaccination could be addressed by improving the timing of pre-travel health care and increasing the acceptance of vaccines. Making pre-travel health care available in primary care settings may be one step to this goal.

In vivo induced antigen technology (IVIAT) is an immuno-screening technique that identifies bacterial antigens expressed during infection and not during standard in vitro culturing conditions. We applied IVIAT to Bacillus anthracis and identified PagA, seven members of a N-acetylmuramoyl-L-alanine amidase autolysin family, three P60 family lipoproteins, two transporters, spore cortex lytic protein SleB, a penicillin binding protein, a putative prophage holin, respiratory nitrate reductase NarG, and three proteins of unknown function. Using quantitative real-time PCR comparing RNA isolated from in vitro cultured B. anthracis to RNA isolated from BALB/c mice infected with virulent Ames strain B. anthracis, we confirmed induced expression in vivo for a subset of B. anthracis genes identified by IVIAT, including L-alanine amidases BA3767, BA4073, and amiA (pXO2-42); the bacteriophage holin gene BA4074; and pagA (pXO1-110). The exogenous addition of two purified putative autolysins identified by IVIAT, N-acetylmuramoyl-L-alanine amidases BA0485 and BA2446, to vegetative B. anthracis cell suspensions induced a species-specific change in bacterial morphology and reduction in viable bacterial cells. Many of the proteins identified in our screen are predicted to affect peptidoglycan re-modeling, and our results support significant cell wall structural remodeling activity during B. anthracis infection. Identification of L-alanine amidases with B. anthracis specificity may suggest new potential therapeutic targets.