High seroprevalence for spotted fever group rickettsiae, is associated with higher temperatures and rural environment in Mbeya region, Southwestern Tanzania.

BACKGROUND: Rickettsioses are endemic in sub-Sahara Africa. Burden of disease, risk factors and transmission are hitherto sparsely described. METHODS: From the EMINI (Evaluating and Monitoring the Impact of New Interventions) population cohort, we randomly selected 1,228 persons above the age of 5 years from the nine participating communities in Mbeya region, Southwestern Tanzania, stratified by age, altitude of residence and ownership of domestic mammals, to conduct a cross-sectional seroprevalence study in. The aim was to estimate the seroprevalence of IgG antibodies against Spotted Fever Group (SFG) rickettsiae and to assess socioeconomic and environmental risk factors. Serology (indirect immunofluorescence) was performed at a dilution of 1:64. RESULTS: SFG-seropositivity in the cohort was found to be 67.9% (range among nine sites: 42.8-91.4%). Multivariable analysis revealed an association with age (prevalence ratio, PR per 10 years: 1.08; 95% CI 1.06-1.10), warmer temperatures (PR per °C: 1.38; 1.11-1.71), male gender (PR 1.08; 1.00-1.16), and low population density (PR per 1.000 persons/km²increase 0.96; 0.94-0.99). At higher elevations, higher cattle density was associated with higher seroprevalence. CONCLUSION: SFG rickettsial infection seems to be common in the more rural population of Mbeya Region. Spread seems to be further limited by temperature and higher elevation. Examination of the contribution of SFG to febrile illnesses seems warranted in a prospective study to estimate the disease burden in the population. This will also allow determination of the causative pathogens.

YmoA negatively controls the expression of insecticidal genes in Yersinia enterocolitica.

Yersinia enterocolitica is toxic towards invertebrates due to the presence of the toxin complex (tc) genes that are activated by the thermolabile regulator TcaR2. In the search for further regulatory factors involved in insecticidal gene expression, the modulator of yersinial virulence, YmoA, was identified to silence all tc genes of the Y. enterocolitica strain W22703 (biovar 2, serovar O:9). Using promoter fusions with the luciferase reporter, we found that the deletion of ymoA results in elevated transcription of tcaR1, tcaR2, tcaA, tcaB, tcaC, tccC1 and tccC2 at both 15 °C and 37 °C. Complementation by episomal ymoA significantly reduced tc gene expression, thus validating the inhibitory activity of YmoA on the production of insecticidal proteins. YmoA contributes to the binding properties of H-NS to the tc promoters by forming a complex with this nucleoid-associated protein, and this complex not only binds to the upstream regions of all tc genes, but also to intragenic sites of tcaA and tcaB that play an important role in controlling the expression of both genes. At low temperature, the intracellular amount of thermostable YmoA is not reduced, but the repressor is less functional. These data point to H-NS/YmoA as an antagonist of the inducer TcaR2.

The insecticidal toxin genes of Yersinia enterocolitica are activated by the thermolabile LTTR-like regulator TcaR2 at low temperatures.

Temperature-dependent activation of bacterial virulence factors at 37°C is well investigated. The molecular mechanism underlying the expression of toxicity determinants at environmental temperatures, however, has not been characterized. The insecticidal activity of Yersinia enterocolitica strain W22703 requires the toxin complex subunit A (TcaA) encoded on the pathogenicity island Tc-PAIYe . Genes tcaA and tcaB encoding this subunit are maximally produced at low temperatures (10-20°C), but repressed at body temperature. Two further insecticidal genes, tcaC (subunit B) and tccC1 (subunit C), are silent at both temperatures. A novel LysR-type transcriptional regulator (LTTR), TcaR2, revealed to be autoregulated and essential for tcaA and tcaB expression in W22703. Expression of tcaR2 is negatively controlled by a second LTTR-like regulator, TcaR1. Gel mobility shift assays confirmed the interaction of TcaR2 with the tcaR2, tcaA and tcaB promoters. The activity of the tcaA promoter in heterologous hosts in the presence of TcaR2 excludes the requirement of additional, Yersinia-specific (co)factors for toxin gene expression. Overproduced TcaR2 protein is shown to be unstable at 37°C, whereas the mRNA of tcaA and tcaR2 is equally stable at low and high temperature. Thus, TcaR2 is a key player in the induction of insecticidal genes in Y. enterocolitica at low temperatures.

Shotgun sequencing of Yersinia enterocolitica strain W22703 (biotype 2, serotype O:9): genomic evidence for oscillation between invertebrates and mammals.

BACKGROUND: Yersinia enterocolitica strains responsible for mild gastroenteritis in humans are very diverse with respect to their metabolic and virulence properties. Strain W22703 (biotype 2, serotype O:9) was recently identified to possess nematocidal and insecticidal activity. To better understand the relationship between pathogenicity towards insects and humans, we compared the W22703 genome with that of the highly pathogenic strain 8081 (biotype1B; serotype O:8), the only Y. enterocolitica strain sequenced so far. RESULTS: We used whole-genome shotgun data to assemble, annotate and analyse the sequence of strain W22703. Numerous factors assumed to contribute to enteric survival and pathogenesis, among them osmoregulated periplasmic glucan, hydrogenases, cobalamin-dependent pathways, iron uptake systems and the Yersinia genome island 1 (YGI-1) involved in tight adherence were identified to be common to the 8081 and W22703 genomes. However, sets of ~550 genes revealed to be specific for each of them in comparison to the other strain. The plasticity zone (PZ) of 142 kb in the W22703 genome carries an ancient flagellar cluster Flg-2 of ~40 kb, but it lacks the pathogenicity island YAPI(Ye), the secretion system ysa and yts1, and other virulence determinants of the 8081 PZ. Its composition underlines the prominent variability of this genome region and demonstrates its contribution to the higher pathogenicity of biotype 1B strains with respect to W22703. A novel type three secretion system of mosaic structure was found in the genome of W22703 that is absent in the sequenced strains of the human pathogenic Yersinia species, but conserved in the genomes of the apathogenic species. We identified several regions of differences in W22703 that mainly code for transporters, regulators, metabolic pathways, and defence factors. CONCLUSION: The W22703 sequence analysis revealed a genome composition distinct from other pathogenic Yersinia enterocolitica strains, thus contributing novel data to the Y. enterocolitica pan-genome. This study also sheds further light on the strategies of this pathogen to cope with its environments.

Development and characterization of rat monoclonal antibodies for N-acylated homoserine lactones.

Quorum sensing (QS) is a communication mechanism between bacteria using diffusible chemical signaling molecules, which are called autoinducers (AI). By detecting the concentration of quorum sensing molecules through binding to a specific receptor protein, bacteria regulate their gene expressions when the concentration of autoinducers and thus the cell density reaches a threshold level. Many Gram-negative bacteria use acylated homoserine lactones (HSLs) as autoinducers. Because of the broad biological functions of HSLs, interest in detection and analysis of HSLs is increasing with a view to their medical, biotechnological, and agricultural applications. In this study, an anti-HSL antibody-based immunochemical detection method has been developed. Four structurally distinct HSL haptens, named HSL1, HSL2, HSL3, and HSL4, have been designed for antibody and assay development. New rat anti-HSL monoclonal antibodies (mAbs) have been produced in-house and characterized with enzyme-linked immunosorbent assays (ELISA), both in the coating antigen and in the enzyme tracer format. Eight mAbs (HSL1-1A5, HSL1-8E1, HSL1/2-2C10, HSL1/2-4H5, HSL4-4C9, HSL4-5E12, HSL4-5H3, and HSL4-6D3) will be presented in this paper. We demonstrate that the anti-HSL mAbs have distinguished sensitivity and selectivity toward HSLs depending upon their chemical structures. The optimized assays are capable of detecting HSLs in the microgram per liter (low micromolar to nanomolar) range. The best IC(50) (test midpoint) was 134 ± 30 µg L(-1) (n = 54) for N-(3-oxodecanoyl)-L-homoserine lactone (3-oxo-C10-HSL) using mAb HSL1/2-2C10 and HSL1-HRP in the enzyme tracer format. In the coating antigen format, the most selective mAb for N-octanoyl-L-homoserine lactone (C8-HSL) was mAb HSL4-4C9. Additionally, anti-HSL mAbs showed higher sensitivity against hydrolyzed HSLs, namely homoserines. These compounds might also occur under certain biological conditions. This study marks the beginning of new ways for quick and cost-effective HSL detection, requiring small sample amounts (less than 1 mL) and little to no sample preparation.

Yersinia enterocolitica infection and tcaA-dependent killing of Caenorhabditis elegans.

Caenorhabditis elegans is a validated model to study bacterial pathogenicity. We report that Yersinia enterocolitica strains W22703 (biovar 2, serovar O:9) and WA314 (biovar 1B, serovar O:8) kill C. elegans when feeding on the pathogens for at least 15 min before transfer to the feeding strain Escherichia coli OP50. The killing by Yersinia enterocolitica requires viable bacteria and, in contrast to that by Yersinia pestis and Yersinia pseudotuberculosis strains, is biofilm independent. The deletion of tcaA encoding an insecticidal toxin resulted in an OP50-like life span of C. elegans, indicating an essential role of TcaA in the nematocidal activity of Y. enterocolitica. TcaA alone is not sufficient for nematocidal activity because E. coli DH5alpha overexpressing TcaA did not result in a reduced C. elegans life span. Spatial-temporal analysis of C. elegans infected with green fluorescent protein-labeled Y. enterocolitica strains showed that Y. enterocolitica colonizes the nematode intestine, leading to an extreme expansion of the intestinal lumen. By low-dose infection with W22703 or DH5alpha followed by transfer to E. coli OP50, proliferation of Y. enterocolitica, but not E. coli, in the intestinal lumen of the nematode was observed. The titer of W22703 cells within the worm increased to over 10(6) per worm 4 days after infection while a significantly lower number of a tcaA knockout mutant was recovered. A strong expression of tcaA was observed during the first 5 days of infection. Y. enterocolitica WA314 (biovar 1B, serovar O:8) mutant strains lacking the yadA, inv, yopE, and irp1 genes known to be important for virulence in mammals were not attenuated or only slightly attenuated in their toxicity toward the nematode, suggesting that these factors do not play a significant role in the colonization and persistence of this pathogen in nematodes. In summary, this study supports the hypothesis that C. elegans is a natural host and nutrient source of Y. enterocolitica.