Viviane Chenal-Francisque

Uncovering Listeria monocytogenes hypervirulence by harnessing its biodiversity

Microbial pathogenesis studies are typically performed with reference strains, thereby overlooking within-species heterogeneity in microbial virulence. Here we integrated human epidemiological and clinical data with bacterial population genomics to harness the biodiversity of the model foodborne pathogen Listeria monocytogenes and decipher the basis of its neural and placental tropisms. Taking advantage of the clonal structure of this bacterial species, we identify clones epidemiologically associated either with food or with human central nervous system (CNS) or maternal-neonatal (MN) listeriosis. The latter clones are also most prevalent in patients without immunosuppressive comorbidities. Strikingly, CNS- and MN-associated clones are hypervirulent in a humanized mouse model of listeriosis. By integrating epidemiological data and comparative genomics, we have uncovered multiple new putative virulence factors and demonstrate experimentally the contribution of the first gene cluster mediating L. monocytogenes neural and placental tropisms. This study illustrates the exceptional power in harnessing microbial biodiversity to identify clinically relevant microbial virulence attributes.

Worldwide Distribution of Major Clones of Listeria monocytogenes

Listeria monocytogenes is worldwide a pathogen, but the geographic distribution of clones remains largely unknown. Genotyping of 300 isolates from the 5 continents and diverse sources showed the existence of few prevalent and globally distributed clones, some of which include previously described epidemic clones. Cosmopolitan distribution indicates the need for genotyping standardization.

Whole genome-based population biology and epidemiological surveillance of Listeria monocytogenes

Listeria monocytogenes (Lm) is a major human foodborne pathogen. Numerous Lm outbreaks have been reported worldwide and associated with a high case fatality rate, reinforcing the need for strongly coordinated surveillance and outbreak control. We developed a universally applicable genome-wide strain genotyping approach and investigated the population diversity of Lm using 1,696 isolates from diverse sources and geographical locations. We define, with unprecedented precision, the population structure of Lm, demonstrate the occurrence of international circulation of strains and reveal the extent of heterogeneity in virulence and stress resistance genomic features among clinical and food isolates. Using historical isolates, we show that the evolutionary rate of Lm from lineage I and lineage II is low (∼2.5 × 10−7 substitutions per site per year, as inferred from the core genome) and that major sublineages (corresponding to so-called ‘epidemic clones’) are estimated to be at least 50–150 years old. This work demonstrates the urgent need to monitor Lm strains at the global level and provides the unified approach needed for global harmonization of Lm genome-based typing and population biology.

Epidemic clones of Listeria monocytogenes are widespread and ancient clonal groups

ABSTRACT The food-borne pathogen Listeria monocytogenes is genetically heterogeneous. Although some clonal groups have been implicated in multiple outbreaks, there is currently no consensus on how “epidemic clones” should be defined. The objectives of this work were to compare the patterns of sequence diversity on two sets of genes that have been widely used to define L. monocytogenes clonal groups: multilocus sequence typing (MLST) and multi-virulence-locus sequence typing (MvLST). Further, we evaluated the diversity within clonal groups by pulsed-field gel electrophoresis (PFGE). Based on 125 isolates of diverse temporal, geographical, and source origins, MLST and MvLST genes (i) had similar patterns of sequence polymorphisms, recombination, and selection, (ii) provided concordant phylogenetic clustering, and (iii) had similar discriminatory power, which was not improved when we combined both data sets. Inclusion of representative strains of previous outbreaks demonstrated the correspondence of epidemic clones with previously recognized MLST clonal complexes. PFGE analysis demonstrated heterogeneity within major clones, most of which were isolated decades before their involvement in outbreaks. We conclude that the “epidemic clone” denominations represent a redundant but largely incomplete nomenclature system for MLST-defined clones, which must be regarded as successful genetic groups that are widely distributed across time and space.

ActA Promotes Listeria monocytogenes Aggregation, Intestinal Colonization and Carriage

Listeria monocytogenes (Lm) is a ubiquitous bacterium able to survive and thrive within the environment and readily colonizes a wide range of substrates, often as a biofilm. It is also a facultative intracellular pathogen, which actively invades diverse hosts and induces listeriosis. So far, these two complementary facets of Lm biology have been studied independently. Here we demonstrate that the major Lm virulence determinant ActA, a PrfA-regulated gene product enabling actin polymerization and thereby promoting its intracellular motility and cell-to-cell spread, is critical for bacterial aggregation and biofilm formation. We show that ActA mediates Lm aggregation via direct ActA-ActA interactions and that the ActA C-terminal region, which is not involved in actin polymerization, is essential for aggregation in vitro. In mice permissive to orally-acquired listeriosis, ActA-mediated Lm aggregation is not observed in infected tissues but occurs in the gut lumen. Strikingly, ActA-dependent aggregating bacteria exhibit an increased ability to persist within the cecum and colon lumen of mice, and are shed in the feces three order of magnitude more efficiently and for twice as long than bacteria unable to aggregate. In conclusion, this study identifies a novel function for ActA and illustrates that in addition to contributing to its dissemination within the host, ActA plays a key role in Lm persistence within the host and in transmission from the host back to the environment.

A horizontally acquired filamentous phage contributes to the pathogenicity of the plague bacillus

Yersinia pestis, the plague bacillus, has an exceptional pathogenicity but the factors responsible for its extreme virulence are still unknown. A genome comparison with its less virulent ancestor Yersinia pseudotuberculosis identified a few Y. pestis‐specific regions acquired after their divergence. One of them potentially encodes a prophage (YpfΦ), similar to filamentous phages associated with virulence in other pathogens. We show here that YpfΦ forms filamentous phage particles infectious for other Y. pestis isolates. Although it was previously suggested that YpfΦ is restricted to the Orientalis branch, our results indicate that it was acquired by the Y. pestis ancestor. In Antiqua and Medievalis strains, YpfΦ genome forms an unstable episome whereas in Orientalis isolates it is stably integrated as tandem repeats. Deletion of the YpfΦ genome does not affect Y. pestis ability to colonize and block the flea proventriculus, but results in an alteration of Y. pestis pathogenicity in mice. Our results show that transformation of Y. pestis from a classical enteropathogen to the highly virulent plague bacillus was accompanied by the acquisition of an unstable filamentous phage. Continued maintenance of YpfΦ despite its high in vitro instability suggests that it confers selective advantages to Y. pestis under natural conditions.

Characterization of the novel Listeria monocytogenes PCR serogrouping profile IVb-v1.

The World Health Organization Collaborating Centre for Listeria (WHOCCL) has developed in 2004 a multiplex PCR assay that separates the 4 major Listeria monocytogenes serovars (1/2a, 1/2b, 1/2c, and 4b) into distinct PCR serogroups. A new PCR profile has been recently identified, constituted of amplified DNA fragments of prs, ORF2819, ORF2110 and lmo0737. Here we characterize 22 L. monocytogenes isolates of the WHOCCL collection with this PCR IVb variant 1 (IVb-v1) profile. The 22 isolates belong to the clinically predominant serovar 4b, exhibit 6 distinct pulsed-field gel electrophoresis ApaI/AscI combined profiles, and belong to 2 unrelated multilocus sequence types, indicating that the novel profile does not correspond to a recent clonal emergence. We have updated the WHOCCL serogroup-related PCR typing scheme to include this new profile.

Efficient tracing of global isolates of Yersinia pestis by restriction fragment length polymorphism analysis using three insertion sequences as probes.

ABSTRACT Yersinia pestis is the etiologic agent of plague, a disease that is transmitted from rodent to rodent and from rodent to humans by fleabites. Multiple copies of three insertion sequences (IS100, IS285, and IS1541) are scattered over the Y. pestis genome. The genomic instability generated by these insertion sequences (IS) creates a polymorphism of the hybridizing restriction fragments (restriction fragment length polymorphism [RFLP]) which can be used to subtype this relatively clonal species. The aim of this work was to evaluate and compare the potential of the three IS-RFLP techniques, individually or in combination, to define clusters of strains according to their focus of origin. The analysis of 61 Y. pestis isolates of worldwide origin indicated that no satisfactory strain clustering was observed with each IS-RFLP used individually. In contrast, the combination of the three IS-RFLP data (3IS-RFLP) resulted in both an efficient strain discrimination (D = 0.999) and a robust clustering of the isolates according to their biovar and geographical origin. This geographical clustering was observed even within the Orientalis group, although these strains had only a short period of time (one century) to diverge from the original clone that spread globally. Therefore, 3IS-RFLP is a technique that may be useful for addressing epidemiological problems and forensic issues. When plague reemerges after several decades of silence in a quiescent focus, it may help in determining whether the disease was reimported or reactivated. It may also be of value to identify the origin of a strain when plague cases appear in a previously plague-free region. Finally, this technique could be useful for the tracing of a Y. pestis isolate that has been used as a biological terrorism threat.

Relationship between bacterial virulence and nucleotide metabolism: a mutation in the adenylate kinase gene renders Yersinia pestis avirulent

Nucleoside monophosphate kinases (NMPKs) are essential catalysts for bacterial growth and multiplication. These enzymes display high primary sequence identities among members of the family Enterobacteriaceae. Yersinia pestis, the causative agent of plague, belongs to this family. However, it was previously shown that its thymidylate kinase (TMPKyp) exhibits biochemical properties significantly different from those of its Escherichia coli counterpart [Chenal-Francisque, Tourneux, Carniel, Christova, Li de la Sierra, Barzu and Gilles (1999) Eur. J. Biochem. 265, 112-119]. In this work, the adenylate kinase (AK) of Y. pestis (AKyp) was characterized. As with TMPKyp, AKyp displayed a lower thermodynamic stability than other studied AKs. Two mutations in AK (Ser129Phe and Pro87Ser), previously shown to induce a thermosensitive growth defect in E. coli, were introduced into AKyp. The recombinant variants had a lower stability than wild-type AKyp and a higher susceptibility to proteolytic digestion. When the Pro87Ser substitution was introduced into the chromosomal adk gene of Y. pestis, growth of the mutant strain was altered at the non-permissive temperature of 37 degree C. In virulence testings, less than 50 colony forming units (CFU) of wild-type Y. pestis killed 100% of the mice upon subcutaneous infection, whereas bacterial loads as high as 1.5 x 10(4) CFU of the adk mutant were unable to kill any animals.

Plague Outbreak in Libya, 2009, Unrelated to Plague in Algeria

After 25 years of no cases of plague, this disease recurred near Tobruk, Libya, in 2009. An epidemiologic investigation identified 5 confirmed cases. We determined ribotypes, Not1 restriction profiles, and IS100 and IS1541 hybridization patterns of strains isolated during this outbreak. We also analyzed strains isolated during the 2003 plague epidemic in Algeria to determine whether there were epidemiologic links between the 2 events. Our results demonstrate unambiguously that neighboring but independent plague foci coexist in Algeria and Libya. They also indicate that these outbreaks were most likely caused by reactivation of organisms in local or regional foci believed to be dormant (Libya) or extinct (Algeria) for decades, rather than by recent importation of Yersinia pestis from distant foci. Environmental factors favorable for plague reemergence might exist in this area and lead to reactivation of organisms in other ancient foci.