Acid stress signals are integrated into the σB-dependent general stress response pathway via the stressosome in the food-borne pathogen Listeria monocytogenes

The general stress response (GSR) in Listeria monocytogenes plays a critical role in the survival of this pathogen in the host gastrointestinal tract. The GSR is regulated by the alternative sigma factor B (σB), whose role in protection against acid stress is well established. However, the mechanisms leading to its activation by low pH are unknown. Here, we investigated the involvement of the stressosome, a sensory organelle, in transducing low pH signals to induce the GSR. Mild acid shock (15 min at pH 5.0) activated σB and conferred protection against a subsequent lethal pH challenge. A mutant strain where the stressosome subunit RsbR1 was present but its remaining paralogues were genetically inactivated retained the ability to induce σB activity at pH 5.0. The role of stressosome phosphorylation in signal transduction was investigated by mutating the putative phosphorylation sites in the core stressosome proteins RsbR1 (rsbR1 T175A, T209A, T241A) and RsbS (rsbS S56A), or in the active site of the stressosome kinase RsbT (rsbT N49A). The rsbS S56A and rsbT N49A mutations abolished the response to low pH. The rsbR1 T175A variant, retained a near-wild type phenotype. The rsbR1 T209A and rsbR1 T241A mutants displayed constitutive σB activity. Mild acid shock upregulates invasion genes and stimulates epithelial cell invasion, effects that were abolished in mutants with an inactive or overactive stressosome. Overall, the results show that the stressosome is required for acid-induced activation of σB in L. monocytogenes. Furthermore, RsbR1 can function independently of its paralogues and that signal transduction requires RsbT-mediated phosphorylation of RsbS on S56 and RsbR1 on T209. These insights shed light on the mechanisms of signal transduction that activate the GSR in L. monocytogenes in response to acidic environments, and highlight the role this sensory process in the early stages of the infectious cycle.

The Different Response to an Acid Shock of Two Salmonella Strains Marks Their Resistance to Thermal Treatments

Microbial cells respond to sub-lethal stresses with several physiological changes to increase their chance of survival. These changes are of high relevance when combined treatments (hurdle technology) are applied during food production, as the cells surviving the first hurdle may have greater resistance to subsequent treatments than untreated cells. In this study, we analyzed if Salmonella develops increased resistance to thermal treatments after the application of an acid shock. We compared the heat resistance of acid-shocked (pH 4.5 achieved with citric acid) Salmonella cells with that of cells maintained at pH 7 (control cells). Thermal treatments were performed between 57.5 and 65°C. We observed a differential response between the two strains studied. Acid-shocked cells of Salmonella Senftenberg exhibited reduced heat resistance, e.g., for a treatment at 60.0°C and pH 7.0 the time required to reduce the population by 3 log cycles was lowered from 10.75 to 1.98min with respect to control cells. Salmonella Enteritidis showed a different response, with acid-shocked cells having similar resistance than untreated cells (the time required to reduce 3 log cycles at 60.0°C and pH 7.0 was 0.30min for control and 0.31min for acid-shock cells). Based on results by differential plating (with or without adding the maximum non-inhibitory concentration of NaCl to the recovery medium), we hypothesize that the differential response between strains can be associated to sub-lethal damage to the cell membrane of S. Senftenberg caused by the acid shock. These results provide evidence that different strains of the same species can respond differently to an acid shock and highlight the relevance of cross-resistances for microbial risk assessment.

Shigella flexneri Diguanylate Cyclases Regulate Virulence

Shigella flexneri is an intracellular human pathogen that invades colonic cells and causes bloody diarrhea. S. flexneri evolved from commensal Escherichia coli , and genome comparisons reveal that S. flexneri has lost approximately 20% of its genes through the process of pathoadaptation, including a disproportionate number of genes associated with the turnover of the nucleotide-based second messenger cyclic di-guanosine monophosphate (c-di-GMP); however, the remaining c-di-GMP turnover enzymes are highly conserved. C-di-GMP regulates many behavioral changes in other bacteria in response to changing environmental conditions, including biofilm formation, but this signaling system has not been examined in S. flexneri . In this study, we expressed VCA0956, a constitutively active c-di-GMP synthesizing diguanylate cyclase (DGC) from Vibrio cholerae , in S. flexneri to determine if virulence phenotypes were regulated by c-di-GMP. We found that expressing VCA0956 in S. flexneri increased c-di-GMP levels, and this corresponds with increased biofilm formation, and reduced acid resistance, host cell invasion, and plaque size. We examined the impact of VCA0956 expression on the S. flexneri transcriptome, and found that genes related to acid resistance were repressed, and this corresponded with decreased survival to acid shock. We also found that individual S. flexneri DGC mutants exhibit reduced biofilm formation, reduced host cell invasion and plaque size, as well as increased resistance to acid shock. This study highlights the importance of c-di-GMP signaling in regulating S. flexneri virulence phenotypes Importance The intracellular human pathogen Shigella causes dysentery, resulting in as many as one million deaths per year. Currently, there is no approved vaccine for the prevention of shigellosis, and the incidence of antimicrobial resistance amongst Shigella species is on the rise. Here, we explore how the widely conserved c-di-GMP bacterial signaling system alters Shigella behaviors associated with pathogenesis. We find that expressing or removing enzymes associated with c-di-GMP synthesis results in changes in Shigella ’s ability to form biofilms, invade host cells, form lesions in host cell monolayers, and resist acid stress.

The impact of acid stress on escherichia coli O157:H7 virulence

Escherichia coli 0157:H7 infection is a leading cause of hemorrhagic colitis, and hemolytic uremic syndrome. Many opportunities for acid stress exposure exist for this food and waterborne pathogen, including gastric acid shock. Yet little is known how this affects E.coli 0157:H7 virulence. The effect of various acid stress protocols on E. coli 0157:H7 survival, verotoxin production, and the ability to adhere to host epithelial cells was examined. Brief acid shock alone at pH 3.0 decreased the host cell adhesion capability by a factor of 4.3-4.8, yet when the acid shock was preceded by adaptation at pH 5.0, a 1.6-3.2 fold enhanced adhesion of surviving organisms to epithelial cells relative to unstressed organisms was observed. However, acid stress did not affect verotoxin production. Pretreatment of acid stressed bacteria with erythromycin eliminated the acid-induced adhesion enhancement, suggesting that protein synthesis is a requirement for the enhanced adhesion observed with acid-adapted acid-shocked E.coli 0157:H7. Real time PCR analysis of locus for enterocyte effacement (LEE)-encoded virulence factors, intimin and EspA, revealed no significant upregulation for the acid stress treatments associated with the increased host cell adhesion. On the contrary, elevated mRNA levels for both intimin and EspA were observed for bacteria subjected to brief acid shock alone even though the host-cell adhesion was significatly decreased with these treatments. These results suggest that complex regulation mechanisms for LEE encoded virulence factors exists and that E. coli 0157:H7 virulence can be enhanced after acid stress through increased adhesion to host epithelial cells.

The impact of acid stress on escherichia coli O157:H7 virulence

Escherichia coli 0157:H7 infection is a leading cause of hemorrhagic colitis, and hemolytic uremic syndrome. Many opportunities for acid stress exposure exist for this food and waterborne pathogen, including gastric acid shock. Yet little is known how this affects E.coli 0157:H7 virulence. The effect of various acid stress protocols on E. coli 0157:H7 survival, verotoxin production, and the ability to adhere to host epithelial cells was examined. Brief acid shock alone at pH 3.0 decreased the host cell adhesion capability by a factor of 4.3-4.8, yet when the acid shock was preceded by adaptation at pH 5.0, a 1.6-3.2 fold enhanced adhesion of surviving organisms to epithelial cells relative to unstressed organisms was observed. However, acid stress did not affect verotoxin production. Pretreatment of acid stressed bacteria with erythromycin eliminated the acid-induced adhesion enhancement, suggesting that protein synthesis is a requirement for the enhanced adhesion observed with acid-adapted acid-shocked E.coli 0157:H7. Real time PCR analysis of locus for enterocyte effacement (LEE)-encoded virulence factors, intimin and EspA, revealed no significant upregulation for the acid stress treatments associated with the increased host cell adhesion. On the contrary, elevated mRNA levels for both intimin and EspA were observed for bacteria subjected to brief acid shock alone even though the host-cell adhesion was significatly decreased with these treatments. These results suggest that complex regulation mechanisms for LEE encoded virulence factors exists and that E. coli 0157:H7 virulence can be enhanced after acid stress through increased adhesion to host epithelial cells.

Advances in yeast autolysis technology - a faster and safer new bioprocess

The yeast autolysis process - an endogenous and irreversible lytic event, which occurs in cells caused by the action of intracellular enzymes, proteases and carbohydrases - is a well-known and an economic process, however, there is a constant risk of serious microbial contamination since there are many nutrients in the broth and this process is slow, favoring the growing of pathogens. The present work comes up with an attempt to accelerate the autolysis of Saccharomyces cerevisiae with focus on the high yield of yeast extract production through a fast, economic and simple technology. The proposed strategy is based on decreasing the pH of the yeast suspension at the beginning of autolysis through an acid shock to activate the cell autolytic system under stressful conditions of temperature and pH. The influence of cell concentration, temperature, time and acid shock at the beginning of the autolysis on yeast extract yields were studied. The best yields of proteins and total solids were observed for autolysis treated with acid shock (H2SO4 10 µL/g of dried yeast and final pH 4.4) at 60 °C (36, 84% of protein and 48, 47% of total solids extracted) and gradual increase of temperature 45 to 60 °C (41.20% of protein and 58.48% of total solids extracted). The shock could increase the speed of the process since the control reached about 30% of extract at 60 °C and the same experiment, however, with acid shock reached more than 43% in 12 h. When considering time in an industrial scale, it could be noted that the time was very important for the productivity as well as avoiding risk of pathogen contamination in autolysis. These results were very relevant for industrial purposes in the production of yeast extract, autolyzed yeast and glucan and mannan.

In-Situ Investigation on Nanoscopic Biomechanics of Streptococcus mutans at Low pH Citric Acid Environments Using an AFM Fluid Cell

Streptococcus mutans (S. mutans) is widely regarded as the main cause of human dental caries via three main virulence factors: adhesion, acidogenicity, and aciduricity. Citric acid is one of the antibiotic agents that can inhibit the virulence capabilities of S. mutans. A full understanding of the acidic resistance mechanisms (ARMs) causing bacteria to thrive in citrate transport is still elusive. We propose atomic force microscopy (AFM) equipped with a fluid cell to study the S. mutans ARMs via surface nanomechanical properties at citric acid pH 3.3, 2.3, and 1.8. Among these treatments, at pH 1.8, the effect of the citric acid shock in cells is demonstrated through a significantly low number of high adhesion zones, and a noticeable reduction in adhesion forces. Consequently, this study paves the way to understand that S. mutans ARMs are associated with the variation of the number of adhesion zones on the cell surface, which is influenced by citrate and proton transport. The results are expected to be useful in developing antibiotics or drugs involving citric acid for dental plaque treatment.

The progress of Type II persisters of Escherichia coli O157:H7 to a non-culturable state during prolonged exposure to antibiotic stress with revival being aided through acid-shock treatment and provision of methyl pyruvate

Persisters are a form of dormancy in bacteria that provide temporary resistance to antibiotics. The following reports on the formation of Escherichia coli O157:H7 E318 Type II persisters from a protracted (8 days) challenge with ampicillin. E. coli O157:H7 followed a multi-phasic die-off pattern with an initial rapid decline (Phase I) of susceptible cells that transitioned to a slower rate representing tolerant cells (Phase II). After 24 h post-antibiotic challenge the E. coli O157: H7 levels remained relatively constant at 2 log CFU/mL (Phase III), but became non-culturable within 8-days (Phase IV). The revival of persisters in Phase III could be achieved by the removal of antibiotic stress although those in Phase IV required an extended incubation period or application of acid-shock. The carbon utilization profile of persister cells was less diverse compared to non-persisters with only methyl pyruvate being utilized from the range tested. Inclusion of methyl pyruvate in TSA revived non-cultural persisters presumably by stimulating metabolism. The results suggested that persisters could be sub-divided into culturable or non-culturable cells with the former representing a transition state to the latter. The study provided insights into how to revive cells from dormancy to aid enumeration and control.

Unveiling the acid stress response of clinical genotypeVibrio vulnificusisolated from the marine environments of Mangaluru coast, India

Gastric acidity is one of the earliest host defences faced by ingested organisms, and successful pathogens need to overcome this hurdle. The objective of this study was the systematic assessment of acid-stress response of Vibrio vulnificus isolated from coastal regions of Mangaluru. Acid-shock experiments were carried out at pH 4.0 and pH 4.5, with different experimental conditions expected to produce a varied acid response. Exposure to mild acid before the acid shock was favourable to the bacteria but was dependent on cell population and pH of the media and was independent of the strains tested. Lysine-dependent acid response was demonstrated with reference to the previously identified lysine decarboxylase system. Additionally, the results showed that inoculation into oysters provided some level of protection against acid stress. Increased expression of lysine/cadaverine genes was observed upon the addition of ground oyster and was confirmed by quantitative real-time PCR. The potential role of ornithine was analyzed with regard to acid stress, but no change in the survival pattern was observed. These findings highlight the physiology of bacteria in acid stress.