Differences in the stability of the plasmids of Yersinia pestis cultures in vitro: impact on virulence

Abstract

Plasmid and chromosomal genes encode determinants of virulence for (Perry & Fetherston 1997, Parkhill et al. 2001, Deng et al. 2002. A 102-kb, unstable chromosomal area (locus pgm) is essential for Y. pestis virulence (Fetherston et al. 1992, Hinnebusch et al. 1996, Buchrieser et al. 1998.Typical strains of Y. pestis harbour three plasmids: pPst (9.5kb), encoding a plasminogen activator protease (Pla) (Sodeinde & Goguen, 1988; pFra (90kb), encoding a capsular protein Fraction 1 (F1) with antiphagocytic activities (Du et al. 2002) and murine toxin (Ymt), required for survival in the flea (Hinnebusch et al. 2002); pYV (70kb), encoding the Yop virulon which comprises both the Yop effectors proteins and the proteins necessary for injecting them into host cells. The Yop virulon enables the bacteria to survive and multiply in the lymphoid tissues of the host (Cornelis 2002). Several insertion sequences (IS100, IS200, IS285) present in the three plasmids favour recombination events and genetic plasticity (Filippov et al. 1995, Parkhill et al. 2001, Deng et al. 2002.However, atypical strains lacking some plasmids have been found in several foci around the world. On the other hand, strains containing extra DNA bands or additional cryptic plasmids have also been found (Filippov et al. 1990, Chu et al. 1998. In vitro, Y. pestis genome is very plastic and several changes have been described: emergence of additional DNA-bands, increasing of plasmid molecular mass, and integration of plasmids into the bacterial chromosome with or without loss of functions (Zsigray et al. 1985, Protsenko et al. 1991. Furthermore, the typical plasmids may be eliminated spontaneously at a high frequency during storage in the laboratory or through successive subcultures (Protsenko et al. 1991).The study of the plasmid content of Y. pestis isolates from the plague foci of Northeast Brazil, stored in the laboratory for several decades, showed that some of them displayed an atypical plasmid profile characterized by the absence of some plasmids or by the presence of extra DNA bands (Leal et al. 1997a, Leal & Almeida 1999, Cavalcanti et al. 2002. The absence of plasmids and the emergence of extra-DNA bands in the Brazilian isolates could also have been produced during storage or handling in the laboratory.To observe possible alterations in plasmids of the cultures in vitro and the impact of the alterations to their pathogenicity, three low subcultured and highly pathogenic Y. pestis isolates were submitted to serial subcultures. Colonies selected after subcultures were analyzed for their plasmid content and the presence of some characteristic genes of each plasmid. LD 50 in mice of the parental strains and derived cultures displaying different plasmid contents were compared. MATERIALS AND METHODSBacteria and culture conditions -The study involved two Brazilian isolates: one old (P. Exu 340, originating from a finger bone marrow in a fatal human case in 1969), and the most recent Brazilian isolate (P. CE 882, originating from a hemoculture from a plagu...

Subtyping Brazilian Yersinia pestis strains by pulsed-field gel electrophoresis

“…Leal-Balbino et al (2004 and Leal et al (2000) observed spontaneous alterations in the genome (plasmid and chromosomal) of Brazilian Y. pestis as a result of prolonged storage and handling in the laboratory. The stability of the PFGE profiles was investigated by the analysis of serial subcultures of three strains.…”

“…Other strains were identified as P.Exu (laboratory location), and sequential number. Three unrelated strains (P.Exu 340, P.Peru 375 and P.CE 882) and 5 derived cultures obtained through multiple serial subcultures of the parental strains (Leal-Balbino et al, 2004) were included to assess the stability of PFGE profiles with subculture. The technique's reproducibility was evaluated by PFGE analysis of the strain P.PB 866 three times.…”

Genetic diversity of Yersinia pestis in Brazil

“…Alternatively, the strains could be inaccurate representatives of the original isolates, and these differences could be due to handling procedures during the prolonged storage of the strains in the laboratory since their isolation (Revazishvili et al, 2008;Vogler et al, 2011). Leal et al (2000) and Leal-Balbino et al (2004, 2006 observed spontaneous changes in the genome (both plasmid and chromosomal) of Brazilian Y. pestis following prolonged storage and laboratory handling. However, analysis of the 12 VNTR loci in the three parental strains and their derived cultures did not reveal changes in the studied regions, confirming their stability as previously observed (Adair et al, 2000;Keim et al, 2000).…”

“…Seventeen strains were isolated from an outbreak in 1986, which involved several municipalities in Planalto da Borborema, Paraíba State (de Almeida et al, 1989). Three unrelated strains (P. CE 882, P. Exu 340 and P. Peru 375) and 11 derived cultures obtained through multiple serial subcultures of the parental strains (Leal-Balbino et al, 2004) were included to assess in vitro VNTR stability. Figure 1 shows the localization of the Brazilian plague foci and origin of the strains.…”

Genome Sequence of the Deep-Rooted Yersinia pestis Strain Angola Reveals New Insights into the Evolution and Pangenome of the Plague Bacterium

“…Enzymatic defects cause incomplete partitioning during replication and decatenation and might result in the dimeric plasmid (14). We speculate that the unique plasmid architecture in Angola helps to guarantee a stable maintenance of both plasmids pMT and pPCP, as these two virulence plasmids are known to be unstable in Y. pestis (44). Indeed, other Pestoides isolates lack one of the two species-specific virulence plasmids (see Fig.…”