Interference competition and high temperature reduce the ‘virulence’ of fig wasps and contribute to stability of a fig-wasp mutualism

Fig trees are pollinated by fig wasps, which also oviposit in female flowers. The wasp larvae gall and eat developing seeds. Although fig trees benefit from allowing wasps to oviposit, because the wasp offspring disperse pollen, figs must prevent wasps from ovipositing in all flowers, or seed production would cease, and the mutualism would go extinct. In Ficus racemosa, we find that syconia (‘figs’) that have few foundresses (ovipositing wasps) are underexploited in the summer (few seeds, few galls, many empty ovules) and are overexploited in the winter (few seeds, many galls, few empty ovules). Conversely, syconia with many foundresses produce intermediate numbers of galls and seeds, regardless of season. We use experiments to explain these patterns, and thus, to explain how this mutualism is maintained. In the hot summer, wasps suffer short lifespans and therefore fail to oviposit in many flowers. In contrast, cooler temperatures in the winter permit longer wasp lifespans, which in turn allows most flowers to be exploited by the wasps. However, even in winter, only in syconia that happen to have few foundresses are most flowers turned into galls. In syconia with higher numbers of foundresses, interference competition reduces foundress lifespans, which reduces the proportion of flowers that are galled. We further show that syconia encourage the entry of multiple foundresses by delaying ostiole closure. Taken together, these factors allow fig trees to reduce galling in the wasp-benign winter and boost galling (and pollination) in the wasp-stressing summer. Interference competition has been shown to reduce virulence in pathogenic bacteria. Our results show that interference also maintains cooperation in a classic, cooperative symbiosis, thus linking theories of virulence and mutualism. More generally, our results reveal how frequency-dependent population regulation can occur in the fig-wasp mutualism, and how a host species can ‘set the rules of the game’ to ensure mutualistic behavior in its symbionts.

Evolution of microbial virulence: the benefits of stress

Although genome sequencing of microbial pathogens has shed light on the evolution of virulence, the drivers of the gain and loss of genes and of pathogenicity islands (gene clusters), which contribute to the emergence of new disease outbreaks, are unclear. Recent experiments with the bean pathogen Pseudomonas syringae pv. phaseolicola illustrate how exposure to resistance mechanisms acts as the driving force for genome reorganization. Here we argue that the antimicrobial conditions generated by host defences can accelerate the generation of genome rearrangements that provide selective advantages to the invading microbe. Similar exposure to environmental stress outside the host could also drive the horizontal gene transfer that has led to the evolution of pathogenicity towards both animals and plants.

Changes in race-specific virulence in pseudomonas syringae pv. phaseolicola are Associated with a chimeric transposable element and rare deletion events in a plasmid-borne pathogenicity island

Virulence for bean and soybean is determined by effector genes in a plasmid-borne pathogenicity island (PAI) in race 7 strain 1449B of Pseudomonas syringae pv. phaseolicola. One of the effector genes, avrPphF, confers either pathogenicity, virulence, or avirulence depending on the plant host and is absent from races 2, 3, 4, 6, and 8 of this pathogen. Analysis of cosmid clones and comparison of DNA sequences showed that the absence of avrPphF from strain 1448A is due to deletion of a continuous 9.5-kb fragment. The remainder of the PAI is well conserved in strains 1448A and 1449B. The left junction of the deleted region consists of a chimeric transposable element generated from the fusion of homologs of IS1492 from Pseudomonas putida and IS1090 from Ralstonia eutropha. The borders of the deletion were conserved in 66 P. syringae pv. phaseolicola strains isolated in different countries and representing the five races lacking avrPphF. However, six strains isolated in Spain had a 10.5-kb deletion that extended 1 kb further from the right junction. The perfect conservation of the 28-nucleotide right repeat of the IS1090 homolog in the two deletion types and in the other 47 insertions of the IS1090 homolog in the 1448A genome strongly suggests that the avrPphF deletions were mediated by the activity of the chimeric mobile element. Our data strongly support a clonal origin for the races of P. syringae pv. phaseolicola lacking avrPphF.

Virulence of entomopathogenic bacteria Xenorhabdus bovienii and Photorhabdus luminescens against Galleria mellonella larvae

Keeping in view the serious health and environmental apprehensions associated with the use of pesticides, entomopathogenic symbiotic bacteria have the potential to supersede pesticides for the management of various pests. Lab experiments were conducted to test the toxicity of two bacteria Xenorhabdus bovienii and Photorhabdus luminescens at different bacterial concentrations against Galleria mellonella larvae and influence of different abiotic factors viz.: substrates, temperatures and moisture levels were ascertained on the efficacy of these bacteria. P. luminescens and X. bovienii caused the maximum mortality (99 and 90%, respectively) at a concentration of 4 x 107 cells/ml. Mortality caused by P. luminescens was significantly higher than that of X. bovienii. Highest mortality was observed on sand as compared to filter paper. A temperature of 30oC and a moisture level of 20 % were found optimum for the maximum mortality.

Galleria mellonella as an infection model for Campylobacter jejuni virulence

Larvae of Galleria mellonella (Greater Wax Moth) have been shown to be susceptible to Campylobacter jejuni infection and our study characterizes this infection model. Following infection with C. jejuni human isolates, bacteria were visible in the haemocoel and gut of challenged larvae, and there was extensive damage to the gut. Bacteria were found in the extracellular and cell-associated fraction in the haemocoel, and it was shown that C. jejuni can survive in insect cells. Finally, we have used the model to screen a further 67 C. jejuni isolates belonging to different MLST types. Isolates belonging to ST257 were the most virulent in the Galleria model, whereas those belonging to ST21 were the least virulent.

Characterisation of Escherichia fergusonii isolates from farm animals using an Escherichia coli virulence gene array and tissue culture adherence assays

Escherichia fergusonii has been associated with a wide variety of intestinal and extra-intestinal infections in both humans and animals but, despite strong circumstantial evidence, the degree to which the organism is responsible for the pathologies identified remains uncertain. Thirty isolates of E fergusonii collected between 2003 and 2004 were screened using an Escherichia coli virulence gene array to test for the presence of homologous virulence genes in E. fergusonii. The iss (increased serum survival) gene was present in 13/30 (43%) of the test strains and the prfB (P-related fimbriae regulatory) and ireA (siderophore receptor IreA) genes were also detected jointly in 3/30 (10%) strains. No known virulence genes were detected in 14/30 (47%) of strains. Following confirmatory PCR and sequence analysis, the E. fergusonii prfB, iss and ireA genes shared a high degree of sequence similarity to their counterparts in E. coli, and a particular resemblance was noted with the E. coli strain APEC O1 pathogenicity island. In tissue culture adherence assays, nine E. fergusonii isolates associated with HEp-2 cells with a 'localised adherence' or 'diffuse adherence' phenotype, and they proved to be moderately invasive. The E fergusonii isolates in this study possess both some phenotypic and genotypic features linked to known pathotypes of E coli, and support existing evidence that strains of E fergusonii may act as an opportunistic pathogens, although their specific virulence factors may need to be explored. Crown Copyright (c) 2008 Published by Elsevier Ltd. All rights reserved.

Periplasmic adaptor protein AcrA has a distinct role in the antibiotic resistance and virulence of Salmonella enterica serovar Typhimurium

Objectives: AcrA can function as the periplasmic adaptor protein (PAP) in several RND tripartite efflux pumps, of which AcrAB-TolC is considered the most important. This system confers innate multiple antibiotic resistance. Disruption of acrB or tolC impairs the ability of Salmonella Typhimurium to colonize and persist in the host. The aim of this study was to investigate the role of AcrA alone in multidrug resistance and pathogenicity. Methods: The acrA gene was inactivated in Salmonella Typhimurium SL1344 by insertion of the aph gene and this mutant complemented with pWKS30acrA. The antimicrobial susceptibility of the mutant to six antibiotics as well as various dyes and detergents was determined. In addition, efflux activity was quantified. The ability of the mutant to adhere to, and invade, tissue culture cells in vitro was measured. Results: Following disruption of acrA, RT-PCR and western blotting confirmed that acrB/AcrB was still expressed when acrA was disrupted. The acrA mutant was hypersusceptible to antibiotics, dyes and detergents. In some cases, lower MICs were seen than for the acrB or tolC mutants. Efflux of the fluorescent dye Hoechst H33342 was less than in wild-type following disruption of acrA. acrA was also required for adherence to, and invasion of, tissue culture cells. Conclusions: Inactivation of acrA conferred a phenotype distinct to that of acrB::aph and tolC::aph. These data indicate a role for AcrA distinct to that of other protein partners in both efflux of substrates and virulence.

Escherichia coli isolates from extraintestinal organs of livestock animals harbour diverse virulence genes and belong to multiple genetic lineages

Escherichia coli, the most common cause of bacteraemia in humans in the UK, can also cause serious diseases in animals. However the population structure, virulence and antimicrobial resistance genes of those from extraintestinal organs of livestock animals are poorly characterised. The aims of this study were to investigate the diversity of these isolates from livestock animals and to understand if there was any correlation between the virulence and antimicrobial resistance genes and the genetic backbone of the bacteria and if these isolates were similar to those isolated from humans. Here 39 E. coli isolates from liver (n=31), spleen (n=5) and blood (n=3) of cattle (n=34), sheep (n=3), chicken (n=1) and pig (n=1) were assigned to 19 serogroups with O8 being the most common (n=7), followed by O101, O20 (both n=3) and O153 (n=2). They belong to 29 multi-locus sequence types, 20 clonal complexes with ST23 (n=7), ST10 (n=6), ST117 and ST155 (both n=3) being most common and were distributed among phylogenetic group A (n=16), B1 (n=12), B2 (n=2) and D (n=9). The pattern of a subset of putative virulence genes was different in almost all isolates. No correlation between serogroups, animal hosts, MLST types, virulence and antimicrobial resistance genes was identified. The distributions of clonal complexes and virulence genes were similar to other extraintestinal or commensal E. coli from humans and other animals, suggesting a zoonotic potential. The diverse and various combinations of virulence genes implied that the infections were caused by different mechanisms and infection control will be challenging.

Effects of repeated cycles of acid challenge and growth on the phenotype and virulence of Salmonella enterica

Aims: The aim of the study was to investigate how stresses like low pH, which may be encountered in farms or food preparation premises, shape populations of Salmonella enterica by the selection of stress-resistant variants. Methods and Results: Stationary-phase cultures of S. enterica serovar Enteritidis and serovar Typhimurium (one strain of each) were exposed to pH 2Æ5 for up to 4 h, followed by growth at pH 7 for 48 h. This process was repeated 15 times in two separate experiments, which increased the acid resistance of the three out of four populations we obtained, by three- to fourfold. Sustainable variants derived from the populations showed changes in colony morphology, expression of SEF17 fimbriae, growth, increased heat resistance and reduced virulence. Conclusions: The study demonstrates that low pH environments can select for populations of S. enterica with persistent phenotypic changes such as increased acid resistance and occasionally increased SEF17 expression and lower virulence. Significance and Impact of the Study: There is a common belief that increased acid resistance coincides with increased virulence. This study demonstrates for the first time that increased acid resistance often impairs virulence and affects the general phenotype of S. enterica.

The CtsR regulator of Listeria monocytogenes contains a variant glycine repeat region that affects piezotolerance, stress resistance, motility and virulence

A spontaneous high hydrostatic pressure (HHP)-tolerant mutant of Listeria monocytogenes ScottA, named AK01, was isolated previously. This mutant was immotile and showed increased resistance to heat, acid and H2O2 compared with the wild type (wt) (Karatzas, K.A.G. and Bennik, M.H.J. 2002 Appl Environ Microbiol 68: 3183–3189). In this study, we conclusively linked the increased HHP and stress tolerance of strain AK01 to a single codon deletion in ctsR (class three stress gene repressor) in a region encoding a highly conserved glycine repeat. CtsR negatively regulates the expression of the clp genes, including clpP, clpE and the clpC operon (encompassing ctsR itself), which belong to the class III heat shock genes. Allelic replacement of the ctsR gene in the wt background with the mutant ctsR gene, designated ctsRΔGly, rendered mutants with phenotypes and protein expression profiles identical to those of strain AK01. The expression levels of CtsR, ClpC and ClpP proteins were significantly higher in ctsRΔGly mutants than in the wt strain, indicative of the CtsRΔGly protein being inactive. Further evidence that the CtsRΔGly protein lacks its repressor function came from the finding that the Clp proteins in the mutant were not further induced upon heat shock, and that HHP tolerance of a ctsR deletion strain was as high as that of a ctsRΔGly mutant. The high HHP tolerance possibly results from the increased expression of the clp genes in the absence of (active) CtsR repressor. Importantly, the strains expressing CtsRΔGly show significantly attenuated virulence compared with the wt strain; however, no indication of disregulation of PrfA in the mutant strains was found. Our data highlight an important regulatory role of the glycine-rich region of CtsR in stress resistance and virulence.