Superinfection drives virulence evolution in experimental populations of bacteria and plasmids

A prominent hypothesis proposes that pathogen virulence evolves in large part due to a trade‐off between infectiousness and damage to hosts. Other explanations emphasize how virulence evolves in response to competition among pathogens within hosts. Given the proliferation of theoretical possibilities, what best predicts how virulence evolves in real biological systems? Here, I show that virulence evolution in experimental populations of bacteria and self‐transmissible plasmids is best explained by within‐host competition. Plasmids evolved to severely reduce the fitness of their hosts even in the absence of uninfected cells. This result is inconsistent with the trade‐off hypothesis, which predicts that under these conditions vertically transmitted pathogens would evolve to be less virulent. Plasmid virulence was strongly correlated with the ability to superinfect cells containing competing plasmid genotypes, suggesting a key role for within‐host competition. When virulent genotypes became common, hosts evolved resistance to plasmid infection. These results show that the trade‐off hypothesis can incorrectly predict virulence evolution when within‐host interactions are neglected. They also show that symbioses between bacteria and plasmids can evolve to be surprisingly antagonistic.     

Virulence plasmids of Shigella bacteria

The contemporary data on the genetical control of invasion process in dysentery causative agent coded by the virulence plasmids of the bacteria are presented. The examples of plasmid genes expression regulated by the genes-regulators located on the chromosome are presented. The mechanism for virulence genes regulation is discussed.     

Genetic Characterization of ExPEC-Like Virulence Plasmids among a Subset of NMEC

Neonatal Meningitis Escherichia coli (NMEC) is one of the most common causes of neonatal bacterial meningitis in the US and elsewhere resulting in mortality or neurologic deficits in survivors. Large plasmids have been shown experimentally to increase the virulence of NMEC in the rat model of neonatal meningitis. Here, 9 ExPEC-like plasmids were isolated from NMEC and sequenced to identify the core and accessory plasmid genes of ExPEC-like virulence plasmids in NMEC and create an expanded plasmid phylogeny. Results showed sequenced virulence plasmids carry a strongly conserved core of genes with predicted functions in five distinct categories including: virulence, metabolism, plasmid stability, mobile elements, and unknown genes. The major functions of virulence-associated and plasmid core genes serve to increase in vivo fitness by adding multiple iron uptake systems to the genetic repertoire to facilitate NMEC’s survival in the host’s low iron environment, and systems to enhance bacterial resistance to host innate immunity. Phylogenetic analysis based on these core plasmid genes showed that at least two lineages of ExPEC-like plasmids could be discerned. Further, virulence plasmids from Avian Pathogenic E. coli and NMEC plasmids could not be differentiated based solely on the genes of the core plasmid genome.     

Informing plasmid compatibility with bacterial hosts using protein-protein interaction data

The compatibility of plasmids with new host cells is significant given their role in spreading antimicrobial resistance (AMR) and virulence factor genes. Evaluating this using in vitro screening is laborious and can be informed by computational analyses of plasmid-host compatibility through rates of protein-protein interactions (PPIs) between plasmid and host cell proteins. We identified large excesses of such PPIs in eight important plasmids, including pOXA-48, using most known bacteria (n = 4363). 23 species had high rates of interactions with four blaOXA-48-positive plasmids. We also identified 48 species with high interaction rates with plasmids common in Escherichia coli. We found a strong association between one plasmid and the fimbrial adhesin operon pil, which could enhance host cell adhesion in aqueous environments. An excess rate of PPIs could be a sign of host-plasmid compatibility, which is important for AMR control given that plasmids like pOXA-48 move between species with ease.     

The virulence plasmids of Salmonella.

Certain Salmonella serovars belonging to subspecies I carry a large, low-copy-number plasmid that contains virulence genes. Virulence plasmids are required to trigger systemic disease; their involvement in the enteric stage of the infection is unclear. Salmonella virulence plasmids are heterogeneous in size (50-90 kb), but all share a 7.8 kb region, spv, required for bacterial multiplication in the reticuloendothelial system. Other loci of the plasmid, such as the fimbrial operon pef, the conjugal transfer gene traT and the enigmatic rck and rsk loci, may play a role in other stages of the infection process. The virulence plasmid of Salmonella typhimurium LT2 is self-transmissible; virulence plasmids from other serovars, such as Salmonella enteritidis and Salmonella choleraesuis, carry incomplete tra operons. The presence of virulence plasmids in host-adapted serovars suggests that virulence plasmid acquisition may have expanded the host range of Salmonella.     

The evolution of a conjugative plasmid and its ability to increase bacterial fitness

Conjugative plasmids are extra-chromosomal DNA elements that are capable of horizontal transmission and are found in many natural isolated bacteria. Although plasmids may carry beneficial genes to their bacterial host, they may also cause a fitness cost. In this work, we studied the evolution of the R1 plasmid and we found that, in spite of the R1 plasmid conferring an initial cost to its host, after 420 generations the cost disappeared in all five independent evolution experiments. In fact, in two of these five experiments evolved conjugative plasmids actually conferred a fitness advantage to their hosts. Furthermore, the relative fitness of the ancestral clone bearing one of the evolved plasmids is significantly higher than both the plasmid-free ancestral cells and the evolved cells carrying the evolved plasmid. Given that the R1 plasmid may spread among different species of enterobacteria, we wondered what the effect of the evolved plasmid would be inside Salmonella enterica cells. We found that the evolved plasmid is also able to dramatically increase the relative fitness of these cells. Our results suggest that even if general usage of antibiotics is halted, conjugative plasmids that have been selected with antibiotics in previous years can still persist among bacterial populations or even invade new strains.     

Plasmids spread very fast in heterogeneous bacterial communities

Conjugative plasmids can mediate gene transfer between bacterial taxa in diverse environments. The ability to donate the F-type conjugative plasmid R1 greatly varies among enteric bacteria due to the interaction of the system that represses sex-pili formations (products of finOP) of plasmids already harbored by a bacterial strain with those of the R1 plasmid. The presence of efficient donors in heterogeneous bacterial populations can accelerate plasmid transfer and can spread by several orders of magnitude. Such donors allow millions of other bacteria to acquire the plasmid in a matter of days whereas, in the absence of such strains, plasmid dissemination would take years. This "amplification effect" could have an impact on the evolution of bacterial pathogens that exist in heterogeneous bacterial communities because conjugative plasmids can carry virulence or antibiotic-resistance genes.     

Impact of Plasmids,Including Those EncodingVirB4/D4 Type IV Secretion Systems,on Salmonella enterica serovar Heidelberg Virulence in Macrophages and Epithelial Cells

Salmonella enterica serovar Heidelberg (S. Heidelberg) can cause foodborne illness in humans following the consumption of contaminated meat and poultry products. Recent studies from our laboratory have demonstrated that certain S. Heidelberg isolated from food-animal sources harbor multiple transmissible plasmids with genes that encode antimicrobial resistance, virulence and a VirB4/D4 type-IV secretion system. This study examines the potential role of these transmissible plasmids in bacterial uptake and survival in intestinal epithelial cells and macrophages, and the molecular basis of host immune system modulation that may be associated with disease progression. A series of transconjugant and transformant strains were developed with different combinations of the plasmids to determine the roles of the individual and combinations of plasmids on virulence. Overall the Salmonella strains containing the VirB/D4 T4SS plasmids entered and survived in epithelial cells and macrophages to a greater degree than those without the plasmid, even though they carried other plasmid types. During entry in macrophages, the VirB/D4 T4SS encoding genes are up-regulated in a time-dependent fashion. When the potential mechanisms for increased virulence were examined using an antibacterial Response PCR Array, the strain containing the T4SS down regulated several host innate immune response genes which likely contributed to the increased uptake and survival within macrophages and epithelial cells.     

The stability region of the large virulence plasmid of Shigella flexneri encodes an efficient postsegregational killing system

The large virulence plasmid pMYSH6000 of Shigella flexneri contains a determinant that is highly effective in stabilizing otherwise unstable plasmids in Escherichia coli. Expression of two small contiguous genes, mvpA and mvpT (formerly termed STBORF1 and STBORF2), was shown to be sufficient for stability. Mutations in mvpT abolished plasmid stability, and plasmids expressing only mvpT killed the cells unless mvpA was supplied from a separate plasmid or from the host chromosome. When replication of a plasmid carrying the minimal mvp region was blocked, growth of the culture stopped after a short lag and virtually all of the surviving cells retained the plasmid. Thus, the mvp system stabilizes by a highly efficient postsegregational killing (PSK) mechanism, with mvpT encoding a cell toxin and mvpA encoding an antidote. The regions that surround the mvp genes in their original context have an inhibitory effect that attenuates plasmid stabilization and PSK. The region encompassing the mvp genes also appears to contain an additional element that can aid propagation of a pSC101-based plasmid under conditions where replication initiation is marginal. However, this appears to be a relatively nonspecific effect of DNA insertion into the plasmid vector.     

Interaction of Shigella sonnei phase-I strains carrying F'- or R386 plasmids with continuous HeLa- and L-line cells

The region of S. sonnei chromosome, located to the left of the gene lac I, has been found to be linked with the capacity of these bacteria for penetrating epithelial cells: this capacity is sharply suppressed in transconjugates carrying plasmids F' which cover the above-mentioned chromosomal region in recipients. The loss of virulence by transconjugates with transferred plasmids F'lac is not linked with the transfer of F factor proper, as those transconjugates which have acquired plasmids F' from E. coli donor strains K = 12 X 363 or F'his 131, not covering the lactose region to the left of the gene lac I, retain their virulence. The transfer of plasmid R 386 having no analogs of bacterial chromosomal genes leads to the loss of virulence due to the oss of the invasive capacity of bacteria.     

Influence of spv plasmid genes group in Salmonella Enteritidis virulence for chickens. I. Occurrence of spv plasmid genes group in Salmonella Enteritidis large virulence plasmid

Many Salmonella Enteritidis virulence factors are encoded by genes localized on plasmids, especially large virulence plasmid, in highly conserved fragment, they create spv plasmid gene group. The aims of realized researches were spv genes occurrence evaluation and composition analysis among Salmonella Enteritidis strains caused infection in chickens. Researches were realized on 107 isolates, where in every cases large virulence plasmid 59 kbp size were detected. Specific nucleotides sequences of spv genes (spvRABCD) were detected in 47.7% of isolates. In the rest of examined bacteria spv genes occurred variably. Most often extreme genes of spv group, like spvR and spvD were absent, what could indicate that factors encoded by them are not most important for Salmonella Enteritidis live and their expressed virulence.     

DISTINGUISHING CAUSES OF VIRULENCE EVOLUTION: REPLY TO ALIZON AND MICHALAKIS

In a recent study of the symbiosis between bacteria and plasmids, the available evidence suggests that experimental evolution of plasmid virulence was primarily driven by within‐host competition caused by superinfection. The data do not exclude the possibility, however, that a trade‐off between virulence and infectious transmission to uninfected bacteria also played a minor role.     

A cooperative virulence plasmid imposes a high fitness cost under conditions that induce pathogenesis

Harbouring a plasmid often imposes a fitness cost on the bacterial host. Motivated by implications for public health, the majority of studies on plasmid cost are focused on elements that impart antibiotic resistance. Plasmids, however, can provide a wide range of ecologically important phenotypes to their bacterial hosts-such as virulence, specialized catabolism and metal resistance. The Agrobacterium tumefaciens tumour-inducing (Ti) plasmid confers both the ability to infect dicotyledonous plants and to catabolize the metabolites that plants produce as a result of being infected. We demonstrate that this virulence and catabolic plasmid imposes a measurable fitness cost on host cells under resource-limiting, but not resource replete, environmental conditions. Additionally, we show that the expression of Ti-plasmid-borne pathogenesis genes necessary to initiate cooperative pathogenesis is extremely costly to the host cell. The benefits of agrobacterial pathogenesis stem from the catabolism of public goods produced by infected host plants. Thus, the virulence-plasmid-dependent costs we demonstrate constitute costs of cooperation typically associated with the ability to garner the benefits of cooperation. Interestingly, genotypes that harbour derived opine catabolic plasmids minimize this trade-off, and are thus able to freeload upon the pathogenesis initiated by other individuals.     

Effect of plasmids on the virulence of Pseudomonas aeruginosa strains in experiments on mice

A comparative study of virulence of P. aeruginosa strains PAO containing and not containing plasmids has been made. A number of plasmids which are present in strains PAO decrease their virulence for mice 3-7 times. The virulence-affecting plasmids considerably differ in their biological properties. Bacterial mutations rpm, selected as mutations stabilizing RP4 plasmid in PAO cells, have also been found to affect virulence of bacteria, decreasing its level several times. The introduction of plasmids into PAO cells carrying mutations rpm is not accompanied by decrease of virulence.     

Design and synthesis of a quintessential self-transmissible IncX1 plasmid, pX1.0

DNA exchange in bacteria via conjugative plasmids is believed to be among the most important contributing factors to the rapid evolution- and diversification rates observed in bacterial species. The IncX1 plasmids are particularly interesting in relation to enteric bacteria, and typically carry genetic loads like antibiotic resistance genes and virulence factors. So far, however, a "pure" version of these molecular parasites, without genetic loads, has yet to be isolated from the environment. Here we report the construction of pX1.0, a fully synthesized IncX1 plasmid capable of horizontal transfer between different enteric bacteria. The designed pX1.0 sequence was derived from the consensus gene content of five IncX1 plasmids and three other, more divergent, members of the same phylogenetic group. The pX1.0 plasmid was shown to replicate stably in E. coli with a plasmid DNA per total DNA ratio corresponding to approximately 3-9 plasmids per chromosome depending on the growth phase of the host. Through conjugation, pX1.0 was able to self-transfer horizontally into an isogenic strain of E. coli as well as into two additional species belonging to the family Enterobacteriaceae. Our results demonstrate the immediate applicability of recent advances made within the field of synthetic biology for designing and constructing DNA systems, previously existing only in silica.     

Complete nucleotide sequence of a virulence plasmid of Salmonella enterica serovar Dublin and its phylogenetic relationship to the virulence plasmids of serovars Choleraesuis, Enteritidis and Typhimurium

The complete nucleotide sequence of pOU1113 (pSDVu), one of the two types of virulence plasmids of Salmonella enterica serovar Dublin, was determined. It contained 80 156 bp with 53.8 mol% G+C content. Approximately 70 genes could be discerned. Compared with pSTV, the virulence plasmid of serovar Typhimurium, pOU1113 was shorter owing to a missing region amounting to c. 10 kb; furthermore, except for a unique 10 849-bp region, the nucleotide as well as deduced amino acid sequences of pOU1113 were nearly identical to the corresponding regions of three S. enterica virulence plasmids, namely pSCV (virulence plasmid of Choleraesuis), pSTV and pSEV (virulence plasmids of Enteritidis), confirming their close phylogenetic relationship. Comparative analysis indicated that these virulence plasmids appeared to have descended by deletion from a relatively large plasmid to smaller ones, with some recombination events occurring over time. From a biological and evolutionary point of view, if the decreasing sizes of pOU1113 and pSCV truly reflect a process in which the virulence plasmid has been shedding unnecessary genes during evolution, our data suggest that some genes in the missing region, such as the pef and tra operons, could have a minimal role in maintaining the survival of the bacteria in their environmental niche.     

Investigating the impact of bisphosphonates and structurally related compounds on bacteria containing conjugative plasmids

Bacterial plasmids propagate through microbial populations via the directed process of conjugative plasmid transfer (CPT). Because conjugative plasmids often encode antibiotic resistance genes and virulence factors, several approaches to inhibit CPT have been described. Bisphosphonates and structurally related compounds (BSRCs) were previously reported to disrupt conjugative transfer of the F (fertility) plasmid in Escherichia coli. We have further investigated the effect of these compounds on the transfer of two additional conjugative plasmids, pCU1 and R100, between E. coli cells. The impact of BSRCs on E. coli survival and plasmid transfer was found to be dependent on the plasmid type, the length of time the E. coli were exposed to the compounds, and the ratio of plasmid donor to plasmid recipient cells. Therefore, these data indicate that BSRCs produce a range of effects on the conjugative transfer of bacterial plasmids in E. coli. Since their impact appears to be plasmid type-dependent, BSRCs are unlikely to be applicable as broad inhibitors of antibiotic resistance propagation.     

The virulence plasmid pWR100 and the repertoire of proteins secreted by the type III secretion apparatus of Shigella flexneri

Bacteria of Shigella spp. are the causative agents of shigellosis. The virulence traits of these pathogens include their ability to enter into epithelial cells and induce apoptosis in macrophages. Expression of these functions requires the Mxi-Spa type III secretion apparatus and the secreted IpaA-D proteins, all of which are encoded by a virulence plasmid. In wild-type strains, the activity of the secretion apparatus is tightly regulated and induced upon contact of bacteria with epithelial cells. To investigate the repertoire of proteins secreted by Shigella flexneri in conditions of active secretion, we determined the N-terminal sequence of 14 proteins that are secreted by a mutant in which secretion was deregulated. Sequencing of the virulence plasmid pWR100 of the S. flexneri strain M90T (serotype 5) has allowed us to identify the genes encoding these secreted proteins and suggests that approximately 25 proteins are secreted by the type III secretion apparatus. Analysis of the G+C content and the relative positions of genes and open reading frames carried by the plasmid, together with information concerning the localization and function of encoded proteins, suggests that pWR100 contains blocks of genes of various origins, some of which were initially carried by four different plasmids.     

Conditional gene vectors regulated in cis

Non-integrating gene vectors, which are stably and extrachromosomally maintained in transduced cells would be perfect tools to support long-term expression of therapeutic genes but preserve the genomic integrity of the cellular host. Small extrachromosomal plasmids share some of these ideal characteristics but are primarily based on virus blueprints. These plasmids are dependent on viral trans-acting factors but they can replicate their DNA molecules in synchrony with the chromosome of the cellular host and segregate to daughter cells in an autonomous fashion. On the basis of the concept of the latent origin of DNA replication of Epstein-Barr virus, oriP, we devised novel derivatives, which exclusively rely on an artificial replication factor for both nuclear retention and replication of plasmid DNA. In addition, an allosteric switch regulates the fate of the plasmid molecules, which are rapidly lost upon addition of doxycycline. Conditional maintenance of these novel plasmid vectors allows the reversible transfer of genetic information into target cells for the first time.