Deletion of toxin–antitoxin systems in the evolution of Shigella sonnei as a host-adapted pathogen

Pathogenic Shigella spp. are the leading cause of bacterial dysentery, with Shigella flexneri and Shigella sonnei accounting for around 90% of cases worldwide. While S. flexneri causes most disease in low-income countries (following ingestion of contaminated food and/or water), S. sonnei predominates in wealthy countries and is mainly spread from person to person. Although both species contain a large virulence plasmid, pINV, that is essential for the organism to cause disease, little is known about its maintenance. Here, using a counterselectable marker within the virulence-encoding region of pINV, we show that the S. sonnei plasmid is less stable than that of S. flexneri, especially at environmental temperatures. GmvAT, a toxin–antitoxin system, is responsible for the difference in stability and is present in pINV from S. flexneri but absent in S. sonnei pINV; GmvT is an acetyltransferase toxin that inhibits protein translation. Loss of GmvAT and a second toxin–antitoxin system, CcdAB, from pINV reduces S. sonnei plasmid stability outside the host, reflecting the host-adapted lifestyle and person-to-person transmission of this species, and hence the striking differences in its epidemiology

Staying out or going in? The interplay between type 3 and type 5 secretion systems in adhesion and invasion of enterobacterial pathogens

Enteric pathogens rely on a variety of toxins, adhesins and other virulence factors to cause infections. Some of the best studied pathogens belong to the Enterobacterales order; these include enteropathogenic and enterohemorrhagic Escherichia coli, Shigella spp., and the enteropathogenic Yersiniae. The pathogenesis of these organisms involves two different secretion systems, a type 3 secretion system (T3SS) and type 5 secretion systems (T5SSs). The T3SS forms a syringe-like structure spanning both bacterial membranes and the host cell plasma membrane that translocates toxic effector proteins into the cytoplasm of the host cell. T5SSs are also known as autotransporters, and they export part of their own polypeptide to the bacterial cell surface where it exerts its function, such as adhesion to host cell receptors. During infection with these enteropathogens, the T3SS and T5SS act in concert to bring about rearrangements of the host cell cytoskeleton, either to invade the cell, confer intracellular motility, evade phagocytosis or produce novel structures to shelter the bacteria. Thus, in these bacteria, not only the T3SS effectors but also T5SS proteins could be considered “cytoskeletoxins” that bring about profound alterations in host cell cytoskeletal dynamics and lead to pathogenic outcomes

Clonal expansion during Staphylococcus aureus infection dynamics reveals the effect of antibiotic intervention

This is the final version of the article. Available from Public Library of Science via the DOI in this recordTo slow the inexorable rise of antibiotic resistance we must understand how drugs impact on pathogenesis and influence the selection of resistant clones. Staphylococcus aureus is an important human pathogen with populations of antibiotic-resistant bacteria in hospitals and the community. Host phagocytes play a crucial role in controlling S. aureus infection, which can lead to a population "bottleneck" whereby clonal expansion of a small fraction of the initial inoculum founds a systemic infection. Such population dynamics may have important consequences on the effect of antibiotic intervention. Low doses of antibiotics have been shown to affect in vitro growth and the generation of resistant mutants over the long term, however whether this has any in vivo relevance is unknown. In this work, the population dynamics of S. aureus pathogenesis were studied in vivo using antibiotic-resistant strains constructed in an isogenic background, coupled with systemic models of infection in both the mouse and zebrafish embryo. Murine experiments revealed unexpected and complex bacterial population kinetics arising from clonal expansion during infection in particular organs. We subsequently elucidated the effect of antibiotic intervention within the host using mixed inocula of resistant and sensitive bacteria. Sub-curative tetracycline doses support the preferential expansion of resistant microorganisms, importantly unrelated to effects on growth rate or de novo resistance acquisition. This novel phenomenon is generic, occurring with methicillin-resistant S. aureus (MRSA) in the presence of β-lactams and with the unrelated human pathogen Pseudomonas aeruginosa. The selection of resistant clones at low antibiotic levels can result in a rapid increase in their prevalence under conditions that would previously not be thought to favor them. Our results have key implications for the design of effective treatment regimes to limit the spread of antimicrobial resistance, where inappropriate usage leading to resistance may reduce the efficacy of life-saving drugs.This work was funded by a Wellcome Trust Project Grant (Reference Number WT089981MA), an EU project: Predicting Antibiotic Resistance (PAR, Reference Number 241476) and from the European Community's Seventh Framework Programme [FP7-PEOPLE-2011-ITN] under grant agreement no. PITN-GA-2011-289209 for the Marie-Curie Initial Training Network FishForPharma. SAR is supported by an MRC Senior Clinical Fellowship (Reference Number: G0701932). Aquarium staff were supported by MRC Centre grant G0700091

Maintenance of the virulence plasmid in Shigella flexneri is influenced by Lon and two functional partitioning systems

Members of the genus Shigella carry a large plasmid, pINV, which is essential for virulence. In Shigella flexneri, pINV harbours three toxin -antitoxin (TA) systems, CcdAB, GmvAT and VapBC that promote vertical transmission of the plasmid. Type II TA systems, such as those on pINV, consist of a toxic protein and protein antitoxin. Selective degradation of the antitoxin by proteases leads to the unopposed action of the toxin once genes encoding a TA system have been lost, such as following failure to inherit a plasmid harbouring a TA system. Here, we investigate the role of proteases in the function of the pINV TA systems and demonstrate that Lon, but not ClpP, is required for their activity during plasmid stability. This provides the first evidence that acetyltransferase family toxin -antitoxin systems, such as GmvAT, can be regulated by Lon. Interestingly, Shigella flexneri pINV also harbours two putative partitioning systems, ParAB and StbAB. We show that both systems are functional for plasmid maintenance although their activity is masked by other systems on pINV . Using a model vector based on the pINV replicon, we observe temperature -dependent differences between the two partitioning systems that contribute to our understanding of the maintenance of virulence in Shigella species

Clonal Expansion during Staphylococcus aureus Infection Dynamics Reveals the Effect of Antibiotic Intervention

To slow the inexorable rise of antibiotic resistance we must understand how drugs impact on pathogenesis and influence the selection of resistant clones. Staphylococcus aureus is an important human pathogen with populations of antibiotic-resistant bacteria in hospitals and the community. Host phagocytes play a crucial role in controlling S. aureus infection, which can lead to a population “bottleneck” whereby clonal expansion of a small fraction of the initial inoculum founds a systemic infection. Such population dynamics may have important consequences on the effect of antibiotic intervention. Low doses of antibiotics have been shown to affect in vitro growth and the generation of resistant mutants over the long term, however whether this has any in vivo relevance is unknown. In this work, the population dynamics of S. aureus pathogenesis were studied in vivo using antibiotic-resistant strains constructed in an isogenic background, coupled with systemic models of infection in both the mouse and zebrafish embryo. Murine experiments revealed unexpected and complex bacterial population kinetics arising from clonal expansion during infection in particular organs. We subsequently elucidated the effect of antibiotic intervention within the host using mixed inocula of resistant and sensitive bacteria. Sub-curative tetracycline doses support the preferential expansion of resistant microorganisms, importantly unrelated to effects on growth rate or de novo resistance acquisition. This novel phenomenon is generic, occurring with methicillin-resistant S. aureus (MRSA) in the presence of β-lactams and with the unrelated human pathogen Pseudomonas aeruginosa. The selection of resistant clones at low antibiotic levels can result in a rapid increase in their prevalence under conditions that would previously not be thought to favor them. Our results have key implications for the design of effective treatment regimes to limit the spread of antimicrobial resistance, where inappropriate usage leading to resistance may reduce the efficacy of life-saving drugs

A privileged intraphagocyte niche is responsible for disseminated infection ofStaphylococcus aureusin a zebrafish model

The innate immune system is the primary defence against the versatile pathogen, Staphylococcus aureus. How this organism is able to avoid immune killing and cause infections is poorly understood. Using an established larval zebrafish infection model, we have shown that overwhelming infection is due to subversion of phagocytes by staphylococci, allowing bacteria to evade killing and found foci of disease. Larval zebrafish coinfected with two S. aureus strains carrying different fluorescent reporter gene fusions (but otherwise isogenic) had bacterial lesions, at the time of host death, containing predominantly one strain. Quantitative data using two marked strains revealed that the strain ratios, during overwhelming infection, were often skewed towards the extremes, with one strain predominating. Infection with passaged bacterial clones revealed the phenomenon not to be due to adventitious mutations acquired by the pathogen. After infection of the host, all bacteria are internalized by phagocytes and the skewing of population ratios is absolutely dependent on the presence of phagocytes. Mathematical modelling of pathogen population dynamics revealed the data patterns are consistent with the hypothesis that a small number of infected phagocytes serve as an intracellular reservoir for S. aureus, which upon release leads to disseminated infection. Strategies to specifically alter neutrophil/macrophage numbers were used to map the potential subpopulation of phagocytes acting as a pathogen reservoir, revealing neutrophils as the likely ‘niche’. Subsequently in a murine sepsis model, S. aureus abscesses in kidneys were also found to be predominantly clonal, therefore likely founded by an individual cell, suggesting a potential mechanism analogous to the zebrafish model with few protected niches. These findings add credence to the argument that S. aureus control regimes should recognize both the intracellular as well as extracellular facets of the S. aureus life cycle

Dually Active Polycation/miRNA Nanoparticles for the Treatment of Fibrosis in Alcohol-Associated Liver Disease

Alcohol-associated liver disease (AALD) is a major cause of liver disorders worldwide. Current treatment options are limited, especially for AALD-associated fibrosis. Promising approaches include RNA interference for miR-155 overexpression in Kupffer cells (KCs), as well as the use of CXCR4 antagonists that inhibit the activation of hepatic stellate cells (HSCs) through the CXCL12/CXCR4 axis. The development of dual-functioning nanoparticles for the effective delivery of antifibrotic RNA together with a CXCR4 inhibitor thus promises to improve the treatment of AALD fibrosis. In this study, cholesterol-modified polymeric CXCR4 inhibitor (Chol-PCX) was synthesized and used to encapsulate anti-miR-155 or non-coding (NC) miRNA in the form of Chol-PCX/miRNA nanoparticles. The results indicate that the nanoparticles induce a significant miR-155 silencing effect both in vitro and in vivo. Treatment with the Chol-PCX/anti-miR-155 particles in a model of moderate alcohol consumption with secondary liver insult resulted in a significant reduction in aminotransferase enzymes as well as collagen content in the liver parenchyma. Overall, our data support the use of Chol-PCX as a carrier for anti-miR-155 for the combined therapeutic inhibition of CXCR4 and miR-155 expression as a way to improve fibrotic damage in the liver

Proteomics Reveal a Concerted Upregulation of Methionine Metabolic Pathway Enzymes, and Downregulation of Carbonic Anhydrase-III, in Betaine Supplemented Ethanol-Fed Rats

We employed a proteomic profiling strategy to examine the effects of ethanol and betaine diet supplementation on major liver protein level changes. Male Wistar rats were fed control, ethanol or betaine supplemented diets for 4 weeks. Livers were removed and liver cytosolic proteins resolved by onedimensional and two-dimensional separation techniques. Significant upregulation of betaine homocysteine methyltransferase-1, methionine adenosyl transferase-1, and glycine N-methyltransferase were the most visually prominent protein changes observed in livers of rats fed the betaine supplemented ethanol diet. We hypothesise that this concerted upregulation of these methionine metabolic pathway enzymes is the protective mechanism by which betaine restores a normal metabolic ratio of liver S-adenosylmethionine to S-adenosylhomocysteine. Ethanol also induced significant downregulation of carbonic anhydrase- III protein levels which was not restored by betaine supplementation. Carbonic anhydrase-III can function to resist oxidative stress, and we therefore hypothesise that carbonic anhydrase-III protein levels compromised by ethanol consumption, contribute to ethanol-induced redox stress

The role of mutation rate variation and genetic diversity in the architecture of human disease

Background We have investigated the role that the mutation rate and the structure of genetic variation at a locus play in determining whether a gene is involved in disease. We predict that the mutation rate and its genetic diversity should be higher in genes associated with disease, unless all genes that could cause disease have already been identified. Results Consistent with our predictions we find that genes associated with Mendelian and complex disease are substantially longer than non-disease genes. However, we find that both Mendelian and complex disease genes are found in regions of the genome with relatively low mutation rates, as inferred from intron divergence between humans and chimpanzees, and they are predicted to have similar rates of non-synonymous mutation as other genes. Finally, we find that disease genes are in regions of significantly elevated genetic diversity, even when variation in the rate of mutation is controlled for. The effect is small nevertheless. Conclusions Our results suggest that gene length contributes to whether a gene is associated with disease. However, the mutation rate and the genetic architecture of the locus appear to play only a minor role in determining whether a gene is associated with disease

RfaH Suppresses Small RNA MicA Inhibition of fimB Expression in Escherichia coli K-12

The phase variation (reversible on-off switching) of the type 1 fimbrial adhesin of Escherichia coli involves a DNA inversion catalyzed by FimB (switching in either direction) or FimE (on-to-off switching). Here, we demonstrate that RfaH activates expression of a FimB-LacZ protein fusion while having a modest inhibitory effect on a comparable fimB-lacZ operon construct and on a FimE-LacZ protein fusion, indicating that RfaH selectively controls fimB expression at the posttranscriptional level. Further work demonstrates that loss of RfaH enables small RNA (sRNA) MicA inhibition of fimB expression even in the absence of exogenous inducing stress. This effect is explained by induction of σE , and hence MicA, in the absence of RfaH. Additional work con- firms that the procaine-dependent induction of micA requires OmpR, as reported previously (A. Coornaert et al., Mol. Microbiol. 76:467–479, 2010, doi:10.1111/j.1365-2958.2010.07115.x), but also demonstrates that RfaH inhibition of fimB transcription is enhanced by procaine independently of OmpR. While the effect of procaine on fimB transcription is shown to be independent of RcsB, it was found to require SlyA, another known regulator of fimB transcription. These results demonstrate a complex role for RfaH as a regulator of fimB expression