Interplay between Yersinia pestis and its flea vector in lipoate metabolism

To thrive, vector-borne pathogens must survive in the vector’s gut. How these pathogens successfully exploit this environment in time and space has not been extensively characterized. Using Yersinia pestis (the plague bacillus) and its flea vector, we developed a bioluminescence-based approach and employed it to investigate the mechanisms of pathogenesis at an unprecedented level of detail. Remarkably, lipoylation of metabolic enzymes, via the biosynthesis and salvage of lipoate, increases the Y. pestis transmission rate by fleas. Interestingly, the salvage pathway’s lipoate/octanoate ligase LplA enhances the first step in lipoate biosynthesis during foregut colonization but not during midgut colonization. Lastly, Y. pestis primarily uses lipoate provided by digestive proteolysis (presumably as lipoyl peptides) rather than free lipoate in blood, which is quickly depleted by the vector. Thus, spatial and temporal factors dictate the bacterium’s lipoylation strategies during an infection, and replenishment of lipoate by digestive proteolysis in the vector might constitute an Achilles’ heel that is exploited by pathogens.Subject terms: Bacterial pathogenesis, Metabolism     

鼠疫耶尔森氏菌环二鸟苷酸代谢及生物被膜形成调控研究进展

鼠疫耶尔森氏菌(Yersinia pestis,以下简称"鼠疫菌")是烈性传染病鼠疫的病原菌,以鼠蚤作为传播媒介。鼠疫菌在其传播媒介鼠蚤的前胃中形成生物被膜从而促进其在宿主间传播。鼠疫菌生物被膜的形成受第二信使分子环二鸟苷酸(c-di-GMP)的正向调控。鼠疫菌中c-di-GMP由二鸟苷酸环化酶(DGC)HmsT和HmsD合成,由磷酸二酯酶(PDE)HmsP降解。文中主要介绍影响鼠疫菌环二鸟苷酸代谢及生物被膜形成的调控因子,并对其作用机制进行讨论和总结。     

Distribution and insecticide resistance of the plague flea Xenopsylla cheopis in Maharashtra State, India

Distributions are reported for commensal rat fleas, predominantly Xenopsylla cheopis (Rothschild), in the State of Maharashtra, India, including the city of Bombay, during 1965-80. The X.cheopis flea index was high in most parts of Maharashtra, but low in Bombay. Rattus rattus Linnaeus is the principal host of X.cheopis, but the host range includes Bandicota bengalensis Gray, Golunda ellioti Gray, Mus musculus Linnaeus, Rattus blandfordi Thomas, R. norvegicus Berkenhout, Suncus caerulaeus Lerr, S. murinus Linnaeus and Tatera indica Hardwicke. X.cheopis was found to have high degrees of resistance to DDT, malathion and fenthion, tolerance to gamma HCH (= gamma BCH) but susceptibility to dieldrin. This insecticide resistance situation may contribute to the high flea indices prevailing in the state, with consequent risks of plague outbreaks. Two other species of rat flea, X.astia Rothschild and X.brasiliensis (Baker) were found to be less common than previously recorded. Their apparent replacement by X.cheopis is tentatively attributed, at least partly, to the selective advantage of insecticide resistance in the latter species.     

Characteristics of digestion of blood of different animals by the flea Xenopsylla cheopis]

Under experimental conditions fleas X. cheopis engorged and assimilated blood of 4 species of rodents, man, pigeon and two species of reptiles. Histological investigations have shown that the structure of food clot, destruction rate of blood cells and general duration of digestion are changed considerably depending on the host's blood. In females the digestion of white mice blood to haematin at 23-24 degrees lasted not over 12 hours, the digestion of blood of hamsters and man - 12 to 20 hours, of guinea pigs and reptiles - 18 to 25 hours and of white rats and pigeons - from 20 to 30 hours. In males the digestion carries out somewhat slower. The above differences in blood digestion can apparently create unequal conditions for pathogenic microbes in the intestine of fleas that affects the preservation and reproduction of the agent in the organisms of these carriers.     

Biological and physico-chemical properties of Yersinia pseudotuberculosis bacterial culture having the fra-operon Yersinia pestis

The biological and physico-chemical properties of cultures of two isogenous recombinant variants of Yersinia pseudotuberculosis were studied. The cell genomes of the cultures are distinguished from one another only by the presence or by the absence of the fra-operon, which is a determined attribute of the plague microbe capsule-forming process. The expression of the attribute is amplified by rising the microbial biomass cultivation temperature and stimulates the decrease in the viability of the bacteria and adaptation potential in vitro. In the warm-blooded owner organism the microbes of the capsule-forming recombinant variant are characterized by the greater residual pathogenicity and immunogenic ability to the experimental plague of the laboratory animals as compared to the reference-variant cells. These specific features could be explained by more expressed colonizing ability of the capsule-forming microbes provided by owner cells' stability to the phagocyte process.     

Analysis of insect toxin complex gene variability of Yersinia pestis and Yersinia pseudotuberculosis strains

The nucleotide sequences of the Tc's insect toxin complex genes have been analyzed in 18 natural strains of the main and non-main subspecies of Yersinia pestis isolated in different natural foci in the Russian Federation, as well as neighboring and more remote countries, as compared to the data on Y. pestis and Y. pseudotuberculosis strains stored in the NCBI GenBank database. The nucleotide sequences of these genes in plague agent strains have been found to be highly conserved, in contrast to those of the pseudotuberculosis agent. The sequences of two genes, tcaC and tccC2, have been found to be almost identical in Y. pestis strains, whereas other three genes (tcaA, tcaB, and tccC1) contain a few mutations, which, however, are not common for all strains of the plague agent. Exceptions are only strains of the Y. pestis biovar orientalis, whose tcaB gene is in a nonfunctional state due to a nucleotide deletion. The results suggest that the formation of the species Y. pestis as an agent of a natural focal infection with a transmissive mechanism has not resulted in degradation of the Tc's complex genes. Instead, these genes are likely to have been altered as the plague agent have been adapting to the new environment.     

苯醚威作用于印鼠客蚤的组织学研究

【目的】通过研究苯醚威对印鼠客蚤 Xenopsylla cheopis (Rothschild,1903)的早 3 龄幼虫和未吸血新羽化成虫的组织学变化,探讨其灭蚤机理,为鼠疫媒介蚤种的防治提供基础资料。【方法】以微量点滴法将苯醚威施药于印鼠客蚤早3龄幼虫和未吸血新羽化成虫,采用组织学、显微摄影及统计学方法观察组织变化。【结果】经苯醚威作用后,印鼠客蚤的早 3 龄幼虫的表皮增厚、卵巢芽生殖细胞萎缩、睾丸芽精原细胞间质减少;未吸血新羽化成虫的睾丸塞消失快、唾液腺细胞破坏严重、中肠上皮细胞萎缩。【结论】(1)苯醚威通过干扰印鼠客蚤幼虫的变态,引起幼虫表皮、生殖芽异常改变,不能发育为成虫而死亡;(2)苯醚威可加速印鼠客蚤新羽化雄性成虫的睾丸塞吸收;(3)苯醚威可破坏印鼠客蚤新羽化成虫的唾液腺细胞,并引起中肠上皮细胞萎缩。     

Cellular fatty acids as chemical markers for differentiation of Yersinia pestis and Yersinia pseudotuberculosis

Aims:  Gas chromatography (GC) was utilized to investigate the cellular fatty acids (CFAs) composition of 141 Yersinia pestis isolates from different plague foci of China, and 20 Yersinia pseudotuberculosis strains as well.
Methods and Results:  The whole cell fatty acid methyl esters (FAMEs) were obtained by saponification, methylation and extraction followed with analysis using a standardized Microbial Identification System (MIS). Y. pestis and Y. pseudotuberculosis strains are quite similar in major CFA profiles, which include 16:0, 17:0 cyclo, 3-OH-14:0, 16:1ω7c and 18:1ω7c, accounting for more than 80% of the total CFAs.
Conclusions:  Yersinia pestis could be easily differentiated from Y. pseudotuberculosis by plotting the ratios of some CFA pairs, i.e.,14:0/18:0 vs 18:1ω7c/18:0, 3-OH-14:0/18:0 vs 18:1ω7c/18:0, 16:1ω7c/18:0 vs 18:1ω7c/18:0, 12:0/18:0 vs 18:1ω7c/18:0 and 12:0 ALDE/18:0 vs 16:1ω7c/18:0 fatty acids.
Significance and Impact of the Study:  In the present study, the normalized Sherlock MIS and Sherlock standard libraries were used to analyse the fatty acid composition of different strains of Y. pestis and Y. pseudotuberculosis . Meanwhile, ratios of certain CFA components were found to serve as chemical markers for differentiating the two closely related bacteria that are difficult to be differentiated by simply comparing CFA profiles based on other researches.     

Pathogenic action of the plague microbe on the flea Xenopsylla cheopis and the ultrastructure of the causative agent at various times of its stay in the vector

Pathology of gastro-intestinal tract of Xenopsylla cheopis fleas infected with plague microbe was determined by means of electron microscopy. Ultrastructure of plague microbe during different periods of its stay in the vector was studied.     

Characterization of outer membrane proteins of Yersinia pestis and Yersinia pseudotuberculosis strains isolated from India

The majority of virulence factors including the 12 Yersinia outer membrane proteins (Yops), 29 Yop secretion proteins (Ysc) and few specific Yop chaperone (Syc) are contributed by the 70 kb LCR middle plasmid of Yersinia pestis. Yersinia pestis isolates recovered during 1994 plague outbreak and rodent surveillance samples of Southern states of India were studied for the presence of important Yops by the conventional procedure of partially purifying outer membrane proteins (Omps) after cultivation in calcium deficient media. Prominent bands numbering 4-5 between 34-42 kDa region corresponding to important Yops were seen in all the isolates as well as in other Yersinia and non-Yersinia species by SDS-PAGE. Western blotting with the polyclonal antisera raised against these Omp preparations revealed few immuno-reactive bands that appeared to be shared among Y. pestis, Y. pseudotruberculosis, Y. enterocolitica, Y. fredrocksenii, Y. intermedia, Y. kristensenii and E. coli. Three recombinant Yop proteins namely, YopM, YopB and LcrV were produced and antisera to these proteins could reveal presence of these Yops in the Y. pestis Omp preparations. In order to further characterize the important Yops among Omps, attempts were made to generate monoclonal antibodies against Omp preparation. Three of the 4 stable reactive clones that were obtained, when tested, had extensive cross-reactions among pathogenic Yersinia species, Y. pestis and Y. pseudotuberculosis isolates, other Yersinia species and the members of Enterobacteriaceae in dot-ELISA and Western blotting. One of the monoclonal antibodies, YP1, exhibited reaction to all the pathogenic Yersinia species and the isolates, with restricted cross-reactivity to Y. intermedia, Y. kristensenii, K. pneumoniae. None of the 4 monoclonal antibodies had reactions with the 3 recombinant Yop proteins. It appears that under low calcium response, the Y. pestis not only activates secretion of Yops but also a large number of other proteins, which as per the present observations are cross-reactive within the family Enterobacteriaceae.