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. Here, we investigated the involvement of the stressosome, a sensory hub, 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 solely present 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 the stressosome kinase RsbT (rsbT-N49A). The rsbS S56A and rsbT N49A mutations abolished the response to low pH. The rsbR1-T209A and rsbR1-T241A mutants displayed constitutive σB activity. Mild acid shock upregulates invasion genes inlAB 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, they show that RsbR1 can function independently of its paralogues and signal transduction requires RsbT-mediated phosphorylation of RsbS on S56 and RsbR1 on T209 but not T175. 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.

In Vitro Evolution of Listeria monocytogenes Reveals Selective Pressure for Loss of SigB and AgrA Function at Different Incubation Temperatures.

The alternative sigma factor B (σB) contributes to the stress tolerance of the foodborne pathogen Listeria monocytogenes by upregulating the general stress response. We previously showed that σB loss-of-function mutations arise frequently in strains of L. monocytogenes and suggested that mild stresses might favor the selection of such mutations. In this study, we performed in vitro evolution experiments (IVEE) where L. monocytogenes was allowed to evolve over 30 days at elevated (42°C) or lower (30°C) incubation temperatures. Isolates purified throughout the IVEE revealed the emergence of sigB operon mutations at 42°C. However, at 30°C, independent alleles in the agr locus arose, resulting in the inactivation of Agr quorum sensing. Colonies of both sigB mutants and agr mutants exhibited a greyer coloration on 7-days-old agar plates than those of the parental strain. Scanning electron microscopy revealed a more complex colony architecture in the wild type than in the mutant strains. sigB mutant strains outcompeted the parental strain at 42°C but not at 30°C, while agr mutant strains showed a small increase in competitive fitness at 30°C. Analysis of 40,080 L. monocytogenes publicly available genome sequences revealed a high occurrence rate of premature stop codons in both the sigB and agrCA loci. An analysis of a local L. monocytogenes strain collection revealed 5 out of 168 strains carrying agrCA alleles. Our results suggest that the loss of σB or Agr confer an increased competitive fitness in some specific conditions and this likely contributes to the emergence of these alleles in strains of L. monocytogenes. IMPORTANCE To withstand environmental aggressions, L. monocytogenes upregulates a large regulon through the action of the alternative sigma factor B (σB). However, σB becomes detrimental for L. monocytogenes growth under mild stresses, which confer a competitive advantage to σB loss-of-function alleles. Temperatures of 42°C, a mild stress, are often employed in mutagenesis protocols of L. monocytogenes and promote the emergence of σB loss-of-function alleles in the sigB operon. In contrast, lower temperatures of 30°C promote the emergence of Agr loss-of-function alleles, a cell-cell communication mechanism in L. monocytogenes. Our findings demonstrate that loss-of-function alleles emerge spontaneously in laboratory-grown strains. These alleles rise in the population as a consequence of the trade-off between growth and survival imposed by the activation of σB in L. monocytogenes. Additionally, our results demonstrate the importance of identifying unwanted hitchhiker mutations in newly constructed mutant strains.

Activation of the Listeria monocytogenes Stressosome in the Intracellular Eukaryotic Environment.

Listeria monocytogenes is a ubiquitous environmental bacterium and intracellular pathogen that responds to stress using predominantly the alternative sigma factor SigB. Stress is sensed by a multiprotein complex, the stressosome, extensively studied in bacteria grown in nutrient media. Following signal perception, the stressosome triggers a phosphorylation cascade that releases SigB from its anti-sigma factor. Whether the stressosome is activated during the intracellular infection is unknown. Here, we analyzed the subcellular distribution of stressosome proteins in L. monocytogenes located inside epithelial cells following their immunodetection in membrane and cytosolic fractions prepared from intracellular bacteria. Unlike bacteria in laboratory media, intracellular bacteria have a large proportion of the core stressosome protein RsbR1 associated with the membrane. However, another core protein, RsbS, is undetectable. Despite the absence of RsbS, a SigB-dependent reporter revealed that SigB activity increases gradually from early (1 h) to late (6 h) postinfection times. We also found that RsbR1 paralogues attenuate the intensity of the SigB response and that the miniprotein Prli42, reported to tether the stressosome to the membrane in response to oxidative stress, plays no role in associating RsbR1 to the membrane of intracellular bacteria. Altogether, these data indicate that, once inside host cells, the L. monocytogenes stressosome may adopt a unique configuration to sense stress and to activate SigB in the intracellular eukaryotic niche. IMPORTANCE The response to stress mediated by the alternative sigma factor SigB has been extensively characterized in Bacillus subtilis and Listeria monocytogenes. These bacteria sense stress using a supramacromolecular complex, the stressosome, which triggers a cascade that releases SigB from its anti-sigma factor. Despite the fact that many structural data on the complex are available and analyses have been performed in mutants lacking components of the stressosome or the signaling cascade, the integration of the stress signal and the dynamics of stressosome proteins following environmental changes remain poorly understood. Our study provides data at the protein level on essential stressosome components and SigB activity when L. monocytogenes, normally a saprophytic bacterium, adapts to an intracellular lifestyle. Our results support activation of the stressosome complex in intracellular bacteria. The apparent loss of the stressosome core protein RsbS in intracellular L. monocytogenes also challenges current models, favoring the idea of a unique stressosome architecture responding to intracellular host cues.

Mild Stress Conditions during Laboratory Culture Promote the Proliferation of Mutations That Negatively Affect Sigma B Activity in Listeria monocytogenes.

In Listeria monocytogenes, the full details of how stress signals are integrated into the σB regulatory pathway are not yet available. To help shed light on this question, we investigated a collection of transposon mutants that were predicted to have compromised activity of the alternative sigma factor B (σB). These mutants were tested for acid tolerance, a trait that is known to be under σB regulation, and they were found to display increased acid sensitivity, similar to a mutant lacking σB (ΔsigB). The transposon insertions were confirmed by whole-genome sequencing, but in each case, the strains were also found to carry a frameshift mutation in the sigB operon. The changes were predicted to result in premature stop codons, with negative consequences for σB activation, independently of the transposon location. Reduced σB activation in these mutants was confirmed. Growth measurements under conditions similar to those used during the construction of the transposon library revealed that the frameshifted sigB operon alleles conferred a growth advantage at higher temperatures, during late exponential phase. Mixed-culture experiments at 42°C demonstrated that the loss of σB activity allowed mutants to take over a population of parental bacteria. Together, our results suggest that mutations affecting σB activity can arise during laboratory culture because of the growth advantage conferred by these mutations under mild stress conditions. The data highlight the significant cost of stress protection in this foodborne pathogen and emphasize the need for whole-genome sequence analysis of newly constructed strains to confirm the expected genotype.IMPORTANCE In the present study, we investigated a collection of Listeria monocytogenes strains that all carried sigB operon mutations. The mutants all had reduced σB activity and were found to have a growth advantage under conditions of mild heat stress (42°C). In mixed cultures, these mutants outcompeted the wild type when mild heat stress was present but not at an optimal growth temperature. An analysis of 22,340 published L. monocytogenes genome sequences found a high rate of premature stop codons present in genes positively regulating σB activity. Together, these findings suggest that the occurrence of mutations that attenuate σB activity can be favored under conditions of mild stress, probably highlighting the burden on cellular resources that stems from deploying the general stress response.

Amino acids other than glutamate affect the expression of the GAD system in Listeria monocytogenes enhancing acid resistance.

The Glutamate Decarboxylase (GAD) system is important for survival of L. monocytogenes and other microorganisms under acidic conditions. Environmental conditions influence the function of the GAD system. Until now, the only conditions known to lead to increased transcription of the GAD system are the stationary phase in rich media and anoxic conditions. Previously, we showed that transcription of the GAD system requires unidentified compounds other than glutamate present in rich media. Following a test looking at various compounds we identified for first time that peptone, tryptone and casamino acids activate the GAD system under oxic conditions suggesting that amino acid(s) other than glutamate and/or peptides are important for the above process. The defined medium, where the GAD system is inactive, once it is supplemented with the above compounds results in an active intracellular and extracellular GAD system and increased acid resistance. Through functional genomics we show that these compounds are required for GadD2 activity and although we previously showed that GadD3 is active part of the intracellular GAD system, the supplementation did not activate this gene. The above is explained by the fact that only gadD2 transcription was upregulated by these compounds while the transcription of gadD1 and gadD3 remained unaffected. Together our results show that the L. monocytogenes GadD2 decarboxylase is activated in the presence of amino acids or peptides other than glutamate, a finding that has important implications for acid tolerance and food safety.

The σB-dependent regulatory sRNA Rli47 represses isoleucine biosynthesis in Listeria monocytogenes through a direct interaction with the ilvA transcript.

The facultative intracellular pathogen Listeria monocytogenes can persist and grow in a diverse range of environmental conditions, both outside and within its mammalian host. The alternative sigma factor Sigma B (σB) plays an important role in this adaptability and is critical for the transition into the host. While some of the functions of the σB regulon in facilitating this transition are understood the role of σB-dependent small regulatory RNAs (sRNAs) remain poorly characterized. In this study, we focused on elucidating the function of Rli47, a σB-dependent sRNA that is highly induced in the intestine and in macrophages. Using a combination of in silico and in vivo approaches, a binding interaction was predicted with the Shine-Dalgarno region of the ilvA mRNA, which encodes threonine deaminase, an enzyme required for branched-chain amino acid biosynthesis. Both ilvA transcript levels and threonine deaminase activity were increased in a deletion mutant lacking the rli47 gene. The Δrli47 mutant displayed a shorter growth lag in isoleucine-depleted growth media relative to the wild-type, and a similar phenotype was also observed in a mutant lacking σB. The impact of the Δrli47 on the global transcription profile of the cell was investigated using RNA-seq, and a significant role for Rli47 in modulating amino acid metabolism was uncovered. Taken together, the data point to a model where Rli47 is responsible for specifically repressing isoleucine biosynthesis as a way to restrict growth under harsh conditions, potentially contributing to the survival of L. monocytogenes in niches both outside and within the mammalian host.

Loss of SigB in Listeria monocytogenes Strains EGD-e and 10403S Confers Hyperresistance to Hydrogen Peroxide in Stationary Phase under Aerobic Conditions.

UNLABELLED: SigB is the main stress gene regulator in Listeria monocytogenes affecting the expression of more than 150 genes and thus contributing to multiple-stress resistance. Despite its clear role in most stresses, its role in oxidative stress is uncertain, as results accompanying the loss of sigB range from hyperresistance to hypersensitivity. Previously, these differences have been attributed to strain variation. In this study, we show conclusively that unlike for all other stresses, loss of sigB results in hyperresistance to H2O2 (more than 8 log CFU ml(-1) compared to the wild type) in aerobically grown stationary-phase cultures of L. monocytogenes strains 10403S and EGD-e. Furthermore, growth at 30°C resulted in higher resistance to oxidative stress than that at 37°C. Oxidative stress resistance seemed to be higher with higher levels of oxygen. Under anaerobic conditions, the loss of SigB in 10403S did not affect survival against H2O2, while in EGD-e, it resulted in a sensitive phenotype. During exponential phase, minor differences occurred, and this result was expected due to the absence of sigB transcription. Catalase tests were performed under all conditions, and stronger catalase results corresponded well with a higher survival rate, underpinning the important role of catalase in this phenotype. Furthermore, we assessed the catalase activity in protein lysates, which corresponded with the catalase tests and survival. In addition, reverse transcription-PCR (RT-PCR) showed no differences in transcription between the wild type and the ΔsigB mutant in various oxidative stress genes. Further investigation of the molecular mechanism behind this phenotype and its possible consequences for the overall phenotype of L. monocytogenes are under way. IMPORTANCE: SigB is the most important stress gene regulator in L. monocytogenes and other Gram-positive bacteria. Its increased expression during stationary phase results in resistance to multiple stresses. However, despite its important role in general stress resistance, its expression is detrimental for the cell in the presence of oxidative stress, as it promotes hypersensitivity against hydrogen peroxide. This peculiar phenotype is an important element of the physiology of L. monocytogenes, and it might help us explain the behavior of this organism in environments where oxidative stress is present.

The impact of different acidic conditions and food substrates on Listeria monocytogenes biofilms development and removal using nanoencapsulated carvacrol.

Listeria monocytogenes biofilms present a significant challenge in the food industry. This study explores the impact of different acidic conditions of culture media and food matrices on the development and removal of biofilms developed on stainless steel surfaces by wild-type (WT) L. monocytogenes strains as well as in two mutant derivatives, ΔsigB and ΔagrA, that have defects in the general stress response and quorum sensing, respectively. Additionally, the study investigates the efficacy of nanoencapsulated carvacrol as an antimicrobial against L. monocytogenes biofilms developed in Tryptic Soy Broth (TSB) culture media acidified to different pH conditions (3.5, 4.5, 5.5, 6.5), and in food substrates (apple juice, strained yogurt, vegetable soup, semi-skimmed milk) having the same pH levels. No biofilm formation was observed for all L. monocytogenes strains at pH levels of 3.5 and 4.5 in both culture media and food substrates. However, at pH 5.5 and 6.5, increased biofilm levels were observed in both the culture media and food substrates, with the WT strain showing significantly higher biofilm formation (3.04-6.05 log CFU cm-2) than the mutant strains (2.30-5.48 log CFU cm-2). For both applications, the nanoencapsulated carvacrol demonstrated more potent antimicrobial activity against biofilms developed at pH 5.5 with 2.23 to 3.61 log reductions, compared to 1.58-2.95 log reductions at pH 6.5, with mutants being more vulnerable in acidic environments. In food substrates, nanoencapsulated carvacrol induced lower log reductions (1.58-2.90) than the ones in TSB (2.02-3.61). These findings provide valuable insights into the impact of different acidic conditions on the development of L. monocytogenes biofilms on stainless steel surfaces and the potential application of nanoencapsulated carvacrol as a biofilm control agent in food processing environments.

A single point mutation in the Listeria monocytogenes ribosomal gene rpsU enables SigB activation independently of the stressosome and the anti-sigma factor antagonist RsbV.

Microbial population heterogeneity leads to different stress responses and growth behavior of individual cells in a population. Previously, a point mutation in the rpsU gene (rpsUG50C) encoding ribosomal protein S21 was identified in a Listeria monocytogenes LO28 variant, which leads to increased multi-stress resistance and a reduced maximum specific growth rate. However, the underlying mechanisms of these phenotypic changes remain unknown. In L. monocytogenes, the alternative sigma factor SigB regulates the general stress response, with its activation controlled by a series of Rsb proteins, including RsbR1 and anti-sigma factor RsbW and its antagonist RsbV. We combined a phenotype and proteomics approach to investigate the acid and heat stress resistance, growth rate, and SigB activation of L. monocytogenes EGDe wild type and the ΔsigB, ΔrsbV, and ΔrsbR1 mutant strains. While the introduction of rpsUG50C in the ΔsigB mutant did not induce a SigB-mediated increase in robustness, the presence of rpsUG50C in the ΔrsbV and the ΔrsbR1 mutants led to SigB activation and concomitant increased robustness, indicating an alternative signaling pathway for the SigB activation in rpsUG50C mutants. Interestingly, all these rpsUG50C mutants exhibited reduced maximum specific growth rates, independent of SigB activation, possibly attributed to compromised ribosomal functioning. In summary, the increased stress resistance in the L. monocytogenes EGDe rpsUG50C mutant results from SigB activation through an unknown mechanism distinct from the classical stressosome and RsbV/RsbW partner switching model. Moreover, the reduced maximum specific growth rate of the EGDe rpsUG50C mutant is likely unrelated to SigB activation and potentially linked to impaired ribosomal function.

Functional γ-Aminobutyrate Shunt in Listeria monocytogenes: role in acid tolerance and succinate biosynthesis.

Listeria monocytogenes, the causative agent of human listeriosis, is known for its ability to withstand severe environmental stresses. The glutamate decarboxylase (GAD) system is one of the principal systems utilized by the bacterium to cope with acid stress, a reaction that produces γ-aminobutyrate (GABA) from glutamate. Recently, we have shown that GABA can accumulate intracellularly under acidic conditions, even under conditions where no extracellular glutamate-GABA exchange is detectable. The GABA shunt, a pathway that metabolizes GABA to succinate, has been described for several other bacterial genera, and the present study sought to determine whether L. monocytogenes has this metabolic capacity, which, if present, could provide a possible route for succinate biosynthesis in L. monocytogenes. Using crude protein extracts from L. monocytogenes EGD-e, we show that this strain exhibits activity for the two main enzyme reactions in the GABA shunt, GABA aminotransferase (GABA-AT) and succinic semialdehyde dehydrogenase (SSDH). Two genes were identified as candidates for encoding these enzyme activities, argD (GABA-AT) and lmo0913 (SSDH). Crude protein extracts prepared from a mutant lacking a functional argD gene significantly reduced GABA-AT activity, while an lmo0913 mutant lost all detectable SSDH activity. The deletion of lmo0913 increased the acid tolerance of EGD-e and showed an increased accumulation of intracellular GABA, suggesting that this pathway plays a significant role in the survival of this pathogen under acidic conditions. This is the first report of such a pathway in the genus Listeria, which highlights an important link between metabolism and acid tolerance and also presents a possible compensatory pathway to partially overcome the incomplete tricarboxylic acid cycle of Listeria.

Trehalose transport occurs via TreB in Listeria monocytogenes and it influences biofilm development and acid resistance.

Listeria monocytogenes is a pathogenic bacterium that can inhabit a diverse range of environmental niches. This is largely attributed to the high proportion of carbohydrate-specific phosphotransferase system (PTS) genes in its genome. Carbohydrates can be assimilated as sources of energy but additionally they can serve as niche-specific cues for L. monocytogenes to shape its global gene expression, in order to cope with anticipated stresses. To examine carbon source utilization among wild L. monocytogenes isolates and to understand underlying molecular mechanisms, a diverse collection of L. monocytogenes strains (n = 168) with whole genome sequence (WGS) data available was screened for the ability to grow in chemically defined media with different carbon sources. The majority of the strains grew in glucose, mannose, fructose, cellobiose, glycerol, trehalose, and sucrose. Maltose, lactose, and rhamnose supported slower growth while ribose did not support any growth. In contrast to other strains, strain1386, which belonged to clonal complex 5 (CC5), was unable to grow on trehalose as a sole carbon source. WGS data revealed that it carried a substitution (N352K) in a putative PTS EIIBC trehalose transporter, TreB, while this asparagine residue is conserved in other strains in this collection. Spontaneous mutants of strain 1386 that could grow in trehalose were found to harbour a reversion of the substitution in TreB. These results provide genetic evidence that TreB is responsible for trehalose uptake and that the N352 residue is essential for TreB activity. Moreover, reversion mutants also restored other unusual phenotypes that strain 1386 displayed, i.e. altered colony morphology, impaired biofilm development, and reduced acid resistance. Transcriptional analysis at stationary phase with buffered BHI media revealed that trehalose metabolism positively influences the transcription of genes encoding amino acid-based acid resistance mechanisms. In summary, our results demonstrated that N352 is key to the function of the sole trehalose transporter TreB in L. monocytogenes and suggest that trehalose metabolism alters physiology to favour biofilm development and acid stress resistance. Moreover, since strain 1386 is among the strains recommended by the European Union Reference Laboratory for conducting food challenge studies in order to determine whether or not L. monocytogenes can grow in food, these findings have important implications for food safety.

A novel RofA-family transcriptional regulator, GadR, controls the development of acid resistance in Listeria monocytogenes.

Stomach acid provides a significant innate barrier to the entry of the food-borne pathogen Listeria monocytogenes into the human gastrointestinal tract. A key determinant of acid resistance in this bacterium is the conserved glutamate decarboxylase system, GadD2 (encoded by the gadT2D2 operon), which helps to maintain the intracellular pH during exposure to gastric acid. In this study, we identified a premature stop codon in a gene located immediately downstream of the gadT2D2 operon that was highly linked to an acid-sensitive phenotype. When this open reading frame was restored through homologous recombination, an acid-resistant phenotype was restored. Through a series of genetic, transcriptomic, and survival experiments, we established that this gene, which we designated gadR, encodes a transcriptional regulator of the gadT2D2 operon. GadR belongs to the RofA family of regulators, primarily found in streptococci, where they are involved in regulating virulence. The data further showed that gadR plays a critical role in the development of acid resistance in response to mild acid exposure, a response that is known as the adaptive acid tolerance response (ATR). A deletion analysis of the gadT2D2 promoter region identified two 18-bp palindromic sequences that are required for the GadR-mediated induction of gadT2D2, suggesting that they act as binding sites for GadR. Overall, this study uncovers a new RofA-like regulator of acid resistance in L. monocytogenes, which plays a significant role in both growth phase-dependent acid resistance and ATR and accounts for previously observed strain-to-strain differences in survival at low pH.IMPORTANCEThe ability to survive the acidic conditions found in the stomach is crucial for the food-borne pathogen Listeria monocytogenes to gain access to the mammalian gastrointestinal tract. Little is currently known about how acid resistance is regulated in this pathogen and why this trait is highly variable between strains. Here, we used comparative genomics to identify a novel RofA-family transcriptional regulator, GadR, that controls the development of acid resistance. The RofA family of regulators was previously found only in a small group of bacterial pathogens, including streptococci, where they regulate virulence properties. We show that gadR encodes the dominant regulator of acid resistance in L. monocytogenes and that its sequence variability accounts for previously observed differences between strains in this trait. Together, these findings significantly advance our understanding of how this important pathogen copes with acid stress and suggest a potential molecular target to aid its control in the food chain.

Manganese uptake mediated by the NRAMP-type transporter MntH is required for acid tolerance in Listeria monocytogenes.

Listeria monocytogenes is a foodborne pathogen that is characterized by its ability to withstand mild stresses (i.e. cold, acid, salt) often encountered in food products or food processing environments. In the previous phenotypic and genotypic characterization of a collection of L. monocytogenes strains, we have identified one strain 1381, originally obtained from EURL-lm, as acid sensitive (reduced survival at pH 2.3) and extremely acid intolerant (no growth at pH 4.9, which supports the growth of most strains). In this study, we investigated the cause of acid intolerance in strain 1381 by isolating and sequencing reversion mutants that were capable of growth at low pH (pH 4.8) to a similar extent as another strain (1380) from the same MLST clonal complex (CC2). Whole genome sequencing showed that a truncation in mntH, which encodes a homologue of an NRAMP (Natural Resistance-Associated Macrophage Protein) type Mn2+ transporter, is responsible for the acid intolerance phenotype observed in strain 1381. However, the mntH truncation alone was not sufficient to explain the acid sensitivity of strain 1381 at lethal pH values as strain 1381R1 (a mntH+ revertant) exhibited similar acid survival to its parental strain at pH 2.3. Further growth experiments demonstrated that Mn2+ (but not Fe2+, Zn2+, Cu2+, Ca2+, or Mg2+) supplementation fully rescues the growth of strain 1381 under low pH conditions, suggesting that a Mn2+ limitation is the likely cause of growth arrest in the mntH- background. Consistent with the important role of Mn2+ in the acid stress response was the finding that mntH and mntB (both encoding Mn2+ transporters) had higher transcription levels following exposure to mild acid stress (pH 5). Taken together, these results provide evidence that MntH-mediated Mn2+ uptake is essential for the growth of L. monocytogenes under low pH conditions. Moreover, since strain 1381 was recommended for conducting food challenge studies by the European Union Reference Laboratory, the use of this strain in evaluating the growth of L. monocytogenes in low pH environments where Mn2+ is scarce should be reconsidered. Furthermore, since it is unknown when strain 1381 acquired the mntH frameshift mutation, the ability of the strains used for challenge studies to grow under food-related stresses needs to be routinely validated.

Characterization of the intracellular glutamate decarboxylase system: analysis of its function, transcription, and role in the acid resistance of various strains of Listeria monocytogenes.

The glutamate decarboxylase (GAD) system is important for the acid resistance of Listeria monocytogenes. We previously showed that under acidic conditions, glutamate (Glt)/γ-aminobutyrate (GABA) antiport is impaired in minimal media but not in rich ones, like brain heart infusion. Here we demonstrate that this behavior is more complex and it is subject to strain and medium variation. Despite the impaired Glt/GABA antiport, cells accumulate intracellular GABA (GABA(i)) as a standard response against acid in any medium, and this occurs in all strains tested. Since these systems can occur independently of one another, we refer to them as the extracellular (GAD(e)) and intracellular (GAD(i)) systems. We show here that GAD(i) contributes to acid resistance since in a ΔgadD1D2 mutant, reduced GABA(i) accumulation coincided with a 3.2-log-unit reduction in survival at pH 3.0 compared to that of wild-type strain LO28. Among 20 different strains, the GAD(i) system was found to remove 23.11% ± 18.87% of the protons removed by the overall GAD system. Furthermore, the GAD(i) system is activated at milder pH values (4.5 to 5.0) than the GAD(e) system (pH 4.0 to 4.5), suggesting that GAD(i) is the more responsive of the two and the first line of defense against acid. Through functional genomics, we found a major role for GadD2 in the function of GAD(i), while that of GadD1 was minor. Furthermore, the transcription of the gad genes in three common reference strains (10403S, LO28, and EGD-e) during an acid challenge correlated well with their relative acid sensitivity. No transcriptional upregulation of the gadT2D2 operon, which is the most important component of the GAD system, was observed, while gadD3 transcription was the highest among all gad genes in all strains. In this study, we present a revised model for the function of the GAD system and highlight the important role of GAD(i) in the acid resistance of L. monocytogenes.

The stressosome is required to transduce low pH signals leading to increased transcription of the amino acid-based acid tolerance mechanisms in Listeria monocytogenes.

Increasing proton concentration in the environment represents a potentially lethal stress for single-celled microorganisms. To survive in an acidifying environment, the foodborne pathogen Listeria monocytogenes quickly activates the alternative sigma factor B (σB), resulting in upregulation of the general stress response (GSR) regulon. Activation of σB is regulated by the stressosome, a multi-protein sensory complex involved in stress detection and signal transduction. In this study, we used L. monocytogenes strains harbouring two stressosome mutants to investigate the role of this complex in triggering expression of known amino acid-based resistance mechanisms in response to low pH. We found that expression of glutamate decarboxylase (gadD3) and arginine and agmatine deiminases (arcA and aguA1, respectively) were upregulated upon acid shock (pH 5 for 15 min) in a stressosome-dependent manner. In contrast, transcription of the arg operons (argGH and argCJBDF), which encode enzymes for the l-arginine biosynthesis pathway, were upregulated upon acid shock in a stressosome-independent manner. Finally, we found that transcription of argR, which encodes a transcriptional regulator of the arc and arg operons, was largely unaffected by acidic shock. Thus, our findings suggest that the stressosome plays a role in activating amino acid-based pH homeostatic mechanisms in L. monocytogenes . Additionally, we show that genes encoding the l-arginine biosynthesis pathway are highly upregulated under acidic conditions, suggesting that intracellular arginine can help withstand environmental acidification in this pathogen.

Rapid, transient, and proportional activation of σ(B) in response to osmotic stress in Listeria monocytogenes.

The osmotic activation of sigma B (σ(B)) in Listeria monocytogenes was studied by monitoring expression of four known σ(B)-dependent genes, opuCA, lmo2230, lmo2085, and sigB. Activation was found to be rapid, transient, and proportional to the magnitude of the osmotic stress applied, features that underpin the adaptability of this pathogen.

Physiological and metabolic diversity in the yeast Kluyveromyces marxianus.

Kluyveromyces marxianus is homothallic hemiascomycete yeast frequently isolated from dairy environments. It possesses phenotypic traits such as enhanced thermotolerance, inulinase production, and rapid growth rate that distinguish it from its closest relative Kluyveromyces lactis. Certain of these traits, notably fermentation of lactose and inulin to ethanol, make this yeast attractive for industrial production of ethanol from inexpensive substrates. There is relatively little known, however, about the diversity in this species, at the genetic, metabolic or physiological levels. This study compared phenotypic traits of 13 K. marxianus strains sourced from two European Culture Collections. A wide variety of responses to thermo, osmotic, and cell wall stress were observed, with some strains showing multi-stress resistance. These traits generally appeared unlinked indicating that, as with other yeasts, multiple resistance/adaptation pathways are present in K. marxianus. The data indicate that it should be possible to identify the molecular basis of traits to facilitate selection or engineering of strains adapted for industrial environments. The loci responsible for mating were also identified by genome sequencing and PCR analysis. It was found that K. marxianus can exist as stable haploid or diploid cells, opening up additional prospects for future strain engineering.

Evolutionary trade-offs between growth and survival: The delicate balance between reproductive success and longevity in bacteria.

All living cells strive to allocate cellular resources in a way that promotes maximal evolutionary fitness. While there are many competing demands for resources the main decision making process centres on whether to proceed with growth and reproduction or to "hunker down" and invest in protection and survival (or to strike an optimal balance between these two processes). The transcriptional programme active at any given time largely determines which of these competing processes is dominant. At the top of the regulatory hierarchy are the sigma factors that commandeer the transcriptional machinery and determine which set of promoters are active at any given time. The regulatory inputs controlling their activity are therefore often highly complex, with multiple layers of regulation, allowing relevant environmental information to produce the most beneficial response. The tension between growth and survival is also evident in the developmental programme necessary to promote biofilm formation, which is typically associated with low growth rates and enhanced long-term survival. Nucleotide second messengers and energy pools (ATP/ADP levels) play critical roles in determining the fate of individual cells. Regulatory small RNAs frequently play important roles in the decision making processes too. In this review we discuss the trade-off that exists between reproduction and persistence in bacteria and discuss some of the recent advances in this fascinating field.

Intracellular accumulation of high levels of gamma-aminobutyrate by Listeria monocytogenes 10403S in response to low pH: uncoupling of gamma-aminobutyrate synthesis from efflux in a chemically defined medium.

It is well established that the glutamate decarboxylase (GAD) system is central to the survival of Listeria monocytogenes at low pH, both in acidic foods and within the mammalian stomach. The accepted model proposes that under acidic conditions extracellular glutamate is transported into the cell in exchange for an intracellular gamma-aminobutyrate (GABA(i)). The glutamate is then decarboxylated to GABA(i), a reaction that consumes a proton, thereby helping to prevent acidification of the cytoplasm. In this study, we show that glutamate supplementation had no influence on either growth rate at pH 5.0 or survival at pH 2.5 when L. monocytogenes 10403S was grown in a chemically defined medium (DM). In response to acidification, cells grown in DM failed to efflux GABA, even when glutamate was added to the medium. In contrast, in brain heart infusion (BHI), the same strain produced significant extracellular GABA (GABA(e)) in response to acidification. In addition, high levels of GABA(i) (>80 mM) were found in the cytoplasm in response to low pH in both growth media. Medium-swap and medium-mixing experiments revealed that the GABA efflux apparatus was nonfunctional in DM, even when glutamate was present. It was also found that the GadT2D2 antiporter/decarboxylase system was transcribed poorly in DM-grown cultures while overexpression of gadD1T1 and gadD3 occurred in response to pH 3.5. Interestingly, BHI-grown cells did not respond with upregulation of any of the GAD system genes when challenged at pH 3.5. The accumulation of GABA(i) in cells grown in DM in the absence of extracellular glutamate indicates that intracellular glutamate is the source of the GABA(i). These results demonstrate that GABA production can be uncoupled from GABA efflux, a finding that alters the way we should view the operation of bacterial GAD systems.

The Vibrio parahaemolyticus Type III Secretion Systems manipulate host cell MAPK for critical steps in pathogenesis.

BACKGROUND: Vibrio parahaemolyticus is a food-borne pathogen causing inflammation of the gastrointestinal epithelium. Pathogenic strains of this bacterium possess two Type III Secretion Systems (TTSS) that deliver effector proteins into host cells. In order to better understand human host cell responses to V. parahaemolyticus, the modulation of Mitogen Activated Protein Kinase (MAPK) activation in epithelial cells by an O3:K6 clinical isolate, RIMD2210633, was investigated. The importance of MAPK activation for the ability of the bacterium to be cytotoxic and to induce secretion of Interleukin-8 (IL-8) was determined. RESULTS: V. parahaemolyticus deployed its TTSS1 to induce activation of the JNK, p38 and ERK MAPK in human epithelial cells. VP1680 was identified as the TTSS1 effector protein responsible for MAPK activation in Caco-2 cells and the activation of JNK and ERK by this protein was important in induction of host cell death. V. parahaemolyticus actively induced IL-8 secretion in a response mediated by TTSS1. A role for VP1680 and for the ERK signalling pathway in the stimulation of IL-8 production in epithelial cells by V. parahaemolyticus was established. Interestingly, TTSS2 inhibited IL-8 mRNA transcription at early stages of interaction between the bacterium and the cell. CONCLUSIONS: This study demonstrated that V. parahaemolyticus activates the three major MAPK signalling pathways in intestinal epithelial cells in a TTSS1-dependent manner that involves the TTSS1 effector VP1680. Furthermore VP1680 and JNK and ERK activation were needed for maximal cytotoxicity of the bacterium. It was shown that V. parahaemolyticus is a strong inducer of IL-8 secretion and that induction reflects a balance between the effects of TTSS1 and TTSS2. Increases in IL-8 secretion were mediated by TTSS1 and VP1680, and augmented by ERK activation. These results shed light on the mechanisms of bacterial pathogenesis mediated by TTSS and suggest significant roles for MAPK signalling during infection with V. parahaemolyticus.