Introduction
In the past 30 years there has been more than a 6-fold increase in the frequency of food outbreaks in the United States resulting from contaminated produce and prompting investigations into the role of environmental reservoirs and food processing techniques on stress conditioning and pathogenicity of infectious bacteria (
Doyle and Erickson, 2008;
Capozzi et al., 2009). Opportunistic enteric pathogens occupy unique and austere niches and must maintain the capacity to react to mercurial environmental conditions and the caustic and highly variable acidic conditions of the gastrointestinal milieu. Environmental strains of enteric bacteria may also respond to acid stress in the form of industrial waste, decomposition of organic matter and the chemical constituents of food preservation. As such, robust response to acid stress is central to survivability in the environment and the host. A key adaptive feature of enteric bacteria involves the rapid induction of physiological and biochemical changes upon exposure to extreme acidity (pH 1–4) after sub-lethal (pH 4–6) preconditioning. This mechanism is referred to as acid habituation (
Goodson and Rowbury, 1989), acid tolerance (
Foster and Hall, 1990) and the acid stress response (ASR); it varies by organism, acid classification (organic vs. inorganic), phase of bacterial growth (logarithmic vs. stationary) and growth media as well as other environmental factors (
Foster, 1991;
Rowbury, 1995;
Lin et al., 1996;
Foster, 1999;
Koutsoumanis et al., 2003). In addition to protection from lethal acidity, ASR has been shown to provide cross protection to other forms of stress (
Rallu et al., 2000;
Frees etal., 2003;
Xie et al., 2004) and may be elicited upon exposure to various environmental cues (
Flahaut et al., 1996;
Frees et al., 2001;
De Angelis and Gobetti, 2004). Further, induction of ASR has been demonstrated to activate additional adaptive behavior phenomena including virulence, biofilm formation, chemotaxis and antibiotic resistance (
Leyer and Johnson, 1992;
Merrell and Camilli, 2002;
Polen et al., 2003;
Maurer et al., 2005;
Butler et al., 2006;
Hayes etal., 2006;
Merrell et al., 2001).
The physiological and biochemical mechanics of acid stress response have frequently been investigated in food borne pathogens such as enterohemorragic
Escherichia coli O157:H7 and
Salmonella enterica Serovar Typhimurium (
S. typhimurium). Recently,
Vibrio cholerae, the waterborne pathogen responsible for Cholera, has emerged as capable of robust ASR. Several studies have investigated ASR in Gram-positive, industrially relevant bacteria, primary pathogens and Archaea (
van de Guchte et al., 2002;
Merrell et al., 2003;
Cotter and Hill, 2003;
Beales, 2004;
Xie et al., 2004;
Ciaramella et al., 2005;
Padan et al., 2005;
Baker-Austin and Dopson, 2007;
Mols etal., 2010); however, reports contextualizing ASR in environmental and clinically derived strains of enteric bacteria are limited. This review will discuss reports on the study of acid stress response and synthesize recent and transformative molecular microbiological investigations of ASR in
E. coli,
S. typhimurium and
V. cholerae in laboratory and environmental conditions and the agricultural phenomena eliciting this response.
Escherichia coli
Escherichia coli is an ubiquitous Gram-negative and rod-shaped bacteria, commonly found in the gastrointestinal tract of warm-blooded animals. Though harmless strains exist, enteric
E. coli is commonly implicated as a food and water borne pathogen. Demonstrating three genetically and physiologically distinct AR strategies, the glucose-repressible oxidative pathway (
Lin et al., 1995;
Lin et al., 1996), glutamate-dependent acid resistance (
GDAR; Hersh et al., 1996) and arginine-dependent ASR (
Lin et al., 1996), this opportunistic pathogen is arguably the “gold-standard” in the study of enteric ASR. In the past 20 years,
E. coli has emerged as an infectious agent when associated with minimally processed foods, thus garnering investigations on stress response and pathogenicity when associated with produce and during food processing (
Capozzi et al., 2009). Arnold etal. (
2001) investigated the impact of the food preservative acetate on expression of ASR genes in enterohemorragic
E. coli (EHEC) O157:H7 reporting>twofold reduction in more than 60 genes; 48 of which are involved in transcription and translational regulation. By contrast, approximately 25 genes were shown have>twofold increased expression; most noteworthy being five
rpoS sigma factors which have been identified as central for response to acid and peroxide stress as well as heat and osmotic shock (
Cheville et al., 1996;
Price et al., 2000;
Nyström et al., 2004). Utilization of alternative sigma factors (i.e. Rpo family) have also been shown to regulate expression of virulence factors as well as genes that enhance both pathogenicity and environmental viability such as colonization and biofilm formation for
E. coli and other enterotypes (
Joelsson et al., 2007;
Dong and Schellhorn, 2010).
Long-term survival in lethal pH is a hallmark of
E. coli ASR with two separate mechanisms that involve amino acid antiporters. The first employs glutamate antiporters and several key decarboxylase enzymes, which maintain internal pH (pH
i;
Hersh et al., 1996). It has been reported that all of these metabolic enzymes must be synthesized for extensive ASR at pH 2; however, either decarboxylase coupled with the glutamate antiporter will provide long-term resistance at pH>2.5 (
Foster, 1999,
2004). Prolonged survival to lethal pH is also facilitated by alteration in membrane phospholipid composition (
Chang and Cronan, 1999) and the subsequent variance in membrane potential (ΔΨ) and increased pH
i (
Richard and Foster; 2004). This durational response to extreme pH also involves synthesis of acid shock proteins that vary temporally during pH exposure (
Foster, 2004) and sequestration of DNA promoter regions (
Choi et al., 2000). Further, it was recently reported that osmolytes such as NaCl, KCl, proline and sucrose facilitate rapid pH
i homeostasis and therefore hasten the recovery of
E. coli from acid shock (
Kitko et al., 2010). The second long-term survival mechanism is linked to arginine-agmatine amino acid antiporters, which participate in extreme acid response (XAR) and are highly conserved phylogenetically (
Iyer et al., 2003). Sun etal. (
2011) recently investigated the role of ATP dependent DNA repair machinery in
E. coli XAR citing marked reduction in cell viability at extreme pH when genes coding for the ATP biosynthesis enzymes (i.e.
purA, purB and
adk) were knocked out. These results suggest that ATP dependent metabolic processes such as select DNA repair systems, contribute to long-term survival at extreme pH. The phenomenon of DNA repair during stress induced mutagenesis was reviewed by Foster (
2007) who reports that in response to environmental cues such as low pH, many alternative sigma factors (i.e. RpoS) moderate increased expression of error prone polymerases (i.e. Pol IV) and downregulate error-correcting enzymes (i.e. RecO/A) to increase mutations that vary progeny genotypes and facilitate survival.
Recent molecular advancements provide for wholesale screening of microbial transcriptomes enabling nuanced understanding of the diverse factors expressed during enteric ASR and the environmental conditions eliciting this response. Maurer et al. (
2005) observed expression variation of approximately 760 genes in response to pH, identifying genes involved in flagellar motility and chemotaxis, oxidative stress and catabolism being variably moderated in response to pH. Further, this study demonstrated the induction of regulons involved in heat shock and oxidative stress response upon exposure to low pH. In an expansive survey of the
E. coli genome, Kang et al. (
2005) suggest a role for the DNA binding protein, FNR, in regulation of nearly 300 genes, including 189 operons, 5% of which are expressed during acid stress under anaerobic conditions. By contrast, Hayes et al. (
2006) report on the impact of oxygen which moderates expression of>260 genes upon exposure to extreme acid with aeration; though core constituents of these enzymes were expressed independent of oxygen. These ASR factors include hydrogenases and enzymes involved in sugar fermentation, antibiotic resistance and cell membrane composition.
In a microarray survey, Tucker et al. (
2002) identified 28 novel genes expressed during GDAR representing functional groups involved in metabolism, cell envelope composition and modification, chaperoning and transcriptional regulation. These findings suggest clustering of nine acid-inducible genes in the
gadA region involved in GDAR. Though the exact role of each of these genes during ASR is unclear, mutations to these factors reduced acid tolerance. Using molecular techniques and bioinformatics, Hommais et al. (
2004) report clustering of
gadE into chromosomal fitness islands which are highly conserved across multiple
E. coli strains. The
in silico analysis suggested preferential denaturing of promoters required for pH homeostasis during acid adaptation. These findings suggest a highly conserved fitness island for ASR in the
E. coli chromosome. This study is complimented by identifying the role of
gadW in activation of
gadE promoters under acidic conditions which resulted in improved colonization capabilities in the infant mouse model (
Tucker et al., 2003). Ma et al. (
2004) characterized the GDAR pathway and identified the novel regulatory factors, EvgA and YdeO, during
gadA activation in exponential and stationary ASR. These factors were identified across several ASR pathways in
E. coli and involve multiple EvgA interactions along inverted repeat promoters in response to acid stress (
Masuda and Church, 2003). Additionally, Ma etal. (
2003) reported on the regulatory role of acid induced GadE/X and W during ASR as they govern expression of glutamine decarboxylases encoded by
gadA and
gadB and the glutamate: γ-aminobutyric acid (GABA) antiporter, GadC. These results suggest that GadE is central to expression of these factors regardless of media or growth conditions, illustrating the complexity of this stress response cascade which involves five regulatory proteins, two sigma factors and multiple feedback loops.
Molecular investigations have resulted in the discovery of additional key factors that directly govern regulatory cascades during
E. coli ASR. Krin et al. (
2010a) identified H-NS as being near the top of the governance hierarchy during glutamate-, arginine- and lysine-dependent acid resistance. Krin et al. (
2010b) also reported that the director of these three ASR strategies is moderated by RcsB. This is facilitated by RcsB-P/GadE complexes that interact with at least one other alternative regulator (i.e. H-NS, HdfR, CadC or AdiY) and subsequently regulate overlapping ASR pathways. It was later shown that RcsB is conserved and required for ASR in
E. coli K12 and O157:H7 and interactions with GadE occur when cells are in logarithmic phase. In stationary phase however, ASR is controlled by RcsB interactions with promoters that are not GadE regulated (
Johnson et al., 2011). The elaborate interactions governing enteric ASR were further elucidated by Zwir et al. (
2005) in a study characterizing the complex regulatory cascade of promoter interactions for the alternative regulatory proteins, PhoP/PhoQ. Using the Gene Promoter Scan technique, this study identified members of the PhoP regulon and characterized previously unknown regulatory interactions, thus mapping the complex network of the transcriptional regulatory cascade involved in moderating ASR. Very recent work has employed systems biology approaches combining molecular and phenotypic analysis combined with computational modeling to identify the transcriptional networks underlying
E. coli ASR; this resulted in identification of the outer membrane family of proteins (Omp) as central to robust response to mild and extreme acid in aerobic and anaerobic conditions (
Stincone et al., 2011).
Studies to assess the role of fermentation and food production on ASR elicitation have been conducted. In one such study, Polen et al. (2003) employed molecular techniques to investigate the impact of exposing
E. coli to the short chain fatty acids propionate and acetate. This study reports increased expression of families of genes involved in flagellar function and biosynthesis (
fli,
fth and
flg), antibiotic resistance (
mar), chemotaxis (
che) and fimbriae morphology (
fim) as well as the reduced expression of carbon uptake and utilization genes (
cst,
gal,
srl,
ebg,
mal and
mgl) based on short-term exposure to propionate and/or acetate. A related study investigates the role of fermentation byproducts (formate and acetate) on
E. coli ASR (
Kirkpatrick et al., 2001) citing increased expression of RpoS and periplasmic transporters in the presence of acetate but reduced expression of this regulatory factor when exposed to formate. A recent investigation of
E. coli K12 and O157:H7 exposure to complex acid cocktails revealed a more robust response on the part of the enterohemorragic strain involving molecular mechanisms not identified in the laboratory strain, thus suggesting improved ASR capability in conditions mimicking those that occur during digestion which subsequently aid in survival in the host GI tract (
King et al., 2010). Bergholz et al. (
2009) identified more than 330 genes, 104 of which are specific to O157:H7, that are upregulated during cultivation of
E. coli in apple juice (pH 3.5), including genes involved in osmotic, acid and oxidative stress. Price et al. (
2000) identified the diet of cattle and the fermentative conditions therein, which subsequently induce RpoS, as central to shedding of viable, acid-adapted
E. coli into the environment thus implicating ASR in carriage and cell survival in the bovine GI tract. Later, Price et al. (
2004) were the first to report on the utilization of select acid response systems based on the chemical profile of the various acidic environments encountered by the pathogen. They demonstrate the crucial role of RpoS for survival in apple cider (pH 3.5) and in cattle intestines, thus prompting efforts to target this regulatory protein in calves colonized by
E. coli O157:H7. Additionally, RpoS has been implicated in the release and survival of other acid adapted enteric pathogens into aquatic environments (
Merrell and Camilli, 2000) and has been shown to regulate multiple stress response pathways of environmentally derived pathogens (
Small et al., 1994;
Cheville et al., 1996;
Nyström, 2004;
Bhagwat et al., 2008). Further, results of a genome-wide expression study suggest that nearly 10% of all
E. coli genes are under the direct or indirect regulatory control of RpoS which governs all cell physiology under non-optimal growth conditions (
Weber et al., 2005).
In an effort to assess the acid adaptive capability of environmentally derived enterohemorragic
E.coli, Bhagwat et al. (
2005) surveyed 82 isolates from 35 countries to examine their GDAR response through traditional laboratory techniques and molecular characterization. Their findings reveal that nearly 40% of the isolates were defective in inducing GDAR under aerobic conditions while approximately 5% of the isolates were defective under both aerobic and fermentative conditions. However, introduction of
rpoS on a low-copy-number plasmid resulted in the restoration of GDAR under aerobic growth in nearly 80% of acid sensitive isolates, emphasizing the significance of this regulatory agent in
E. coli ASR. Further, environments where GDAR positive EHEC isolates originated may represent conditioning reservoirs for acid resistance and pathogenicity.
Salmonella enterica Serovar Typhimurium
Salmonella enterica Serovar Typhimurium is a Gram-negative, neutralophile commonly found in the intestines of animals such as birds, reptiles and humans making it another model organism in the study of enteric ASR. It is also found in contaminated and polluted pond water and within macrophage phagolysozomes; and, it has been shown to colonize a broad varietal of fruits, vegetables, nuts and poultry products resulting in its distinction as the most common foodborne pathogen (
Foster and Hall, 1990;
Faucher et al., 2006;
Hanning et al., 2009). The acid adaptive capabilities of
S. typhimurium vary according to log or stationary growth and involve synthesis of early and late stage acid shock proteins (ASPs) which moderate cell surface features (i.e. hydrophobicity and outer membrane porins), cytoplasmic pH and macromolecular repair (
Foster, 1991;
Foster, 1993;
Bearson et al., 1998). The various stages of
S. typhimurium acid resistance are governed by the regulatory proteins RpoS, Fur and PhoP/PhoQ, with each responding to a wide array of environmental cues and regulating separate subsets of ASR (
Foster, 1991;
Foster, 1993;
Bader et al., 2003). Collectively these regulatory factors provide cross protection to numerous forms of environmental stress such as temperature, oxidative damage, salinity and antimicrobial agents (
Leyer and Johnson, 1993;
Foster and Spector, 1995;
Bader et al., 2003;
Greenacre et al., 2006) and enhance the expression of multiple virulence and colonization factors (
Foster and Hall, 1990;
Wilmes-Riesenberg et al., 1997;
Dong and Schellhorn, 2010).
S. typhimurium has been investigated with regards to ASR variability based on population growth phase and acid classification. During logarithmic growth,
S. typhimurium ASR involves induction of the alternative sigma factor σ
s (
rpoS) which moderate multiple ASPs in response to short chain fatty acids but play a reduced role during inorganic acid exposure (
Bearson et al., 1998). During stationary phase, ASR is determined in an RpoS-independent manner, which involves induction of outer membrane porins (Omp) in response to environmental cues such as pH and salinity;
V. cholerae has been shown to respond in a similar manner (
Foster, 1999). As is reported in other enteric pathogens, this pathway involves the regulatory factors PhoP/PhoQ which differentiate between acids (inorganic vs. organic), Mg concentrations and enhance expression of virulence factors when exposed to moderate levels of organic acids and when cultivated in mammalian phagosomes (
Bearson et al., 1998;
Prost et al., 2007); phenomena conserved in
Salmonella enterica Serovar Typhi in response to host immunity (
Faucher et al., 2006).
Regarding the impact of food processing on elicitation of enteric ASR, adaptation in inorganic acid has been demonstrated as sufficient for
S. typhimurium and several other
Salmonella species to remain viable and virulent for up to two months in organically acidic cheeses (
Leyer and Johnson, 1992). Further,
S. typhimurium cultivated in acidic conditions typical of food processing displayed robust acid tolerance at a lower pH (pH 4.5) than was observed for
Listeria monocytogenes (pH 5.5) or
E. coli O157:H7 (pH 5.0), suggesting enhanced ASR induction capability, and reduced sensitivity to environmental pH, compared to the common food pathogens
L. monocytogenes and
E. coli (
Koutsoumanis and Sofos, 2004). Acid habituation of
S. typhimurium during food production has been shown to provide cross protection to environmental stress such as heat, salt and lactoperoxidases, and confers tolerance to cell wall destroying agents and antibiotics (
Leyer and Johnson, 1992). However, short-term exposure of
S. typhimurium to the food preservative lactic acid was recently shown to reduce resistance to hydrogen peroxide via downregulation of the OxyR regulon which imparts protection to oxidative stress (
Greenacre et al., 2006).
The interactions between enteric pathogens and the organisms they colonize may play a significant role in elicitation of ASR and was recently investigated by Bhagwat (
2006) in a report on the linkage between plant stress response and acid adaptation in
S. typhimurium. In this study,
S. typhimurium cultivated on cut or damaged plant displayed elevated percent population survival upon subsequent exposure to lethal pH in laboratory conditions (pH2, 37°C, 2 h). In addition to the mildly acidic conditions of these wounded fruits and vegetables, apoplastic fluid from decomposing plants colonized by soft rot pathogens may induce acid resistance of
S. typhimurium (
Nachin and Barras, 2000). The antimicrobial peptides that are part of a wounded plants immune response result in upregulation of ASR factors in
S. typhimurium’s
sap operon which enhance survival upon subsequent exposure to the mammalian gut (
Parra-Lopez etal., 1993;
López-Solanilla et al., 1998,
2001). This pathogen has been shown to respond to cues from bacterial community constituents on spoiled plant biomass via acyl-homoserine lactones (AHLs) produced by the bacterial community (
Ahmer, 2004), prompting the hypothesis that this inter-cellular communication is upregulating determinants of human pathogen growth on plants during decomposition (
Brandl, 2006). Additionally, these interactions have been shown to enhance population densities of
S. typhimurium in wounded fruits and vegetables when co-occurring with other bacterial species (
Wade and Beuchat, 2003) or proteolytic yeasts (
Wade et al., 2003); with the latter being implicated as a transmission vector of enterics into plant tissue (
Richards and Beuchat, 2005). It was recently demonstrated that
S. typhimurium associated with the food vacuoles of the protist
Tetrahymena, alter the expression of approximately 1000 and 1200 genes compared to water and culture broth respectively, including enhanced expression of alternative electron acceptors and factors involved in anaerobic respiration (
Rehfuss et al., 2011). Further, expression of arginine-dependent ASR factors (i.e. AdiA, AdiY) were increased in
S. typhimurium cells within the food vacuole, suggesting that elicitation of ASR confers resistance to digestion from the protist, provides survival advantage upon egestion and may represent an ASR pre-conditioning reservoir in the contamination cycle of
S. typhimurium.
The role of plants as disease vectors during outbreaks of Salmonellosis has been realized since the 1990s when the shift in food contamination became clearly linked to irrigation and plant health (
Hanning et al., 2009). Since then, there has been renewed interest in the role of aquatic reservoirs and the bacterial load thereof, in conditioning enteric pathogens and displacing them to fruit and vegetable products in various agricultural settings. Freshwater systems have commonly been a source of irrigation (
Assadian et al., 1999;
Garcia et al., 2001) and assessing density, virulence and elicitation of stress response of
Salmonella in these aquatic reservoirs has become essential to risk management and infection reduction (
Baudart et al., 2000). In one such study employing general media and media selecting for, and differentiating between, enteric pathogens,
Salmonella was shown to be proportionally dominant in samples taken from beaches and freshwater reservoirs in North-eastern Spain (
Polo et al., 1998). It has also been demonstrated, via traditional culturing techniques, that
Salmonella is a dominant component of the cultivable bacterial community in the water column and in sediment systems impacted by point and non-point sources of pollution in a coastal Mediterranean system; with residence in marine sediments being central to its long-term viability in these aquatic reservoirs (
Baudart et al., 2000). Cultivation in environmental conditions has been shown to increase expression of key virulence factors, with variability in pH being cited as the environmental parameter regulating virulence in samples taken from regions along the Rio Grande in southern Texas (
Nutt et al., 2003).
Vibrio cholerae
Vibrio cholerae is the highly motile, Gram-negative, curve-shaped bacteria responsible for the gastrointestinal disease Cholera. This microorganism follows the oral route of infection, survives the low pH conditions of the stomach (pH 1–3) and colonizes the small intestine (pH 6–8). Here, it produces the cholera toxin (CT) that results in the loss of copious volumes of fluid from the host, and returns virulent strains to the aquatic environment. The mystery of how this environmentally ubiquitous bacteria, which is categorized as acid-sensitive but is capable of causing gastrointestinal disease in epidemic proportions, has been deciphered by the discovery that
V. cholerae mounts a robust ASR when pre-conditioned at sub-lethal pH (
Merrell and Camilli, 2002). In general,
V. cholerae ASR involves overlapping responses to inorganic and organic acids, increased expression of>60 and decrease in>50 protein species with functions including transcriptional regulation, cell wall maintenance, catabolism, colonization, DNA repair, Na
+ homeostasis and membrane transport; collectively resulting in reduced sensitivity to acidic conditions and a greater capacity to cause harm in the mammalian host (
Merrell and Camilli, 1999,
2000,
2002;
Peterson, 2002;
Reidl and Klose, 2002). Acid adapted
V. cholerae display increased colonization capabilities and competitive advantage; thus requiring a reduced infective dose compared to non-adapted cells (
Merrell and Camilli, 1999). This enhanced pathogenicity results from greater reproductive potential within the host, as opposed to increased expression of virulence factors such as cholera toxin (
ctx) or toxin co-regulated pili (
tcp) (
Angelichio et al., 2004).
The first groundbreaking studies on
V. cholerae ASR employed signature tagged mutagenesis (STM) to identify novel factors involved in acid adaptation (
Chiang and Mekalanos, 1998;
Merrell and Camilli, 1999,
2002). Merrell and Camilli (
1999) were the first to identify the ASR factor CadA in
V. cholerae, which moderates pH
i via lysine consumption in the bacterial cytoplasm. Since this study, the Cad family of proteins has been implicated in moderating pH
i during
S. typhimurium ASR (
Greenacre et al., 2006) and in the aquatic pathogen
V. vulnificus (
Rhee et al., 2004). Screening large pools of virulence attenuated
V. cholerae resulted in identification of 9 novel ASR factors involved in sodium and potassium homeostasis, cell wall maintenance, DNA repair and protein synthesis (
Merrell et al., 2002a). Acid-adapted
V. cholerae with enhanced expression of these ASR factors were shown to be more competitive mutants and non-adapted cells when challenged to the suckling mouse model.
As is observed among enteric species,
V. cholerae ASR involves activation of the Rpo family of transcriptional regulators which are central to quorum sensing (
Joelsson etal., 2007), expression of virulence factors (
Dong and Schellhorn, 2010) and secretion of outer membrane vesicles that improve colonization of the intestinal milieu (
Song et al., 2008). Additionally, the Rpo family moderates biofilm formation and expression of the virulence pathogenicity island (VPI) during low and high cell density via the transcriptional regulators, AphA and HapR (
Zhu and Mekalanos, 2003;
Kovacikova et al., 2010;
Rutherford et al., 2011). These factors have been shown to govern ASR based on environmental conditions, with AphB moderating virulence in response to low pH and anaerobiosis (
Kovacikova etal., 2010) and HapR governing protease production, motility, biofilm formation and expression of virulence factors
in vivo (
Zhu and Mekalanos, 2003).
Investigations into the role of the outer membrane porins (Omp) in
V. cholerae ASR have revealed important interactions between this family of proteins and the intercellular regulatory agent’s central to gene expression. Kovacikova and Skorupski (
2002) report on the role of the Omp family in moderating the regulatory protein RpoE and enhancing infectivity and colonization capabilities of
V. cholerae and
E. coli. Mathur et al. (
2007) demonstrate that OmpU acts as an environmental sensor in the signal transduction pathway which regulates the alternative sigma factor, σE and confers resistance to antibiotics such as polymixin B and human gut derived bacteriocidal proteins. Mathur and Waldor (
2004) report on OmpU moderated ToxR expression as essential for antimicrobial resistance and the expression of virulence factors. Though not acid shock proteins per se, as Omp expression is not moderated exclusively by acid, this family of porins has been demonstrated as responsive to physiochemical signals such as bile and organic acids and responsible for regulating alternative sigma factors central to
V.cholerae ASR (
Li et al., 2000;
Provenzano and Klose, 2000;
Merrell and Camilli, 2000;
Matson et al., 2007). Recent molecular investigations detecting genes involved in acid stress response (i.e.
ompU and
toxR), as well as other virulence and regulatory factors in
V. cholerae, report a high degree of conservation of these factors among genetically diverse strains of
V. cholerae isolated from aquatic environments within countries where it is endemic (
Zo et al., 2009;
Goel and Jiang, 2010). The conservation of such stress factors is imperative for environmental survival which is emphasized by the finding that
V. cholerae from pH stressed environments is capable of growth at rates comparable to the natural assemblages in alkaline lake water (pH 7.8–9.1;
Kirschner etal., 2008).
The Tox family (ToxT, ToxR and ToxS) has been shown central to transcriptional regulation for myriad factors involved in
V. cholerae ASR. These regulators are moderated by the Omp family of proteins which stimulate transcription in response to environmental cues by binding
toxbox regions upstream of promoter elements (
Withey and DiRita, 2005,
2006). Subsequently, tox proteins govern a cascade of ASR factors involved in virulence (
ctx, tcp), colonization (
msh), antimicrobial resistance (
mex), cellular metabolism (
gal,
lac, mrs) and DNA repair (
rec); they are activated by an array of metabolic intermediates, regulatory messengers and environmental conditions such as temperature, salinity and pH (
Merrell and Camilli, 2002;
Peterson, 2002;
Reidl and Klose, 2002;
Tischler and Camilli, 2004;
Abuaita and Withey, 2009).
Several studies have aimed to characterize the expression profile of ASR genes as
V. cholerae transitions between the host GI tract and aquatic reservoirs.
In vivo results suggest that
V. cholerae that has passed through the host GI tract demonstrates enhanced virulence upon re-introduction to the host (
Merrell and Camilli, 2002;
Reidl and Klose, 2002). This increased infectivity is facilitated by reduced expression of chemotaxis factors such as CheW-1; a phenotype maintained in the aquatic environment and only elicited post-infection (
Butler et al., 2006;
Merrell et al., 2001). Schild et al. (
2007) investigated
V. cholerae in transit through the host and report increased expression of six unique genes in the latter stage of infection which play limited roles during infection, are critical for survival upon introduction to the aquatic reservoir and are expressed prior to exiting the host. In this study, viability of
V. cholerae expressing late stage genes after transfer to various environments (i.e. rice water stools, pond water and nutrient broth) was monitored and resulted in identification of multiple factors functioning in transcriptional regulation, substrate level phosphorylation and nutrient transport; all of which improve the fitness of
V. cholerae upon introduction to environmental reservoirs.
Investigations of
V. cholerae transitioning between the aquatic environment and the mammalian host have revealed the role of quorum sensing induced biofilm formation as implicit in surviving the host defenses and inducing infection upon entering the GI tract (
Kamruzzaman et al., 2010). Biofilm formation has been shown to involve activation of the regulatory protein, HapR upon exposure to bile acid and is central for
V. cholerae survival in the aquatic environment (
Zhu and Mekalanos, 2003). Further, Faruque et al. (
2006) report that biofilm aggregated
V. cholerae display enhanced viability in the environment after passage through the mammalian host, suggesting a role of ASR for survival in aquatic reservoirs. This study is complimented by the finding that biofilm formation in the environment results in a hyper-infective phenotype upon introduction to the host GI tract (
Tamayo et al., 2010). Beyhan et al. (
2006) report increased expression of genes involved in biofilm formation (
vps), extracellular protein secretion (
eps) and mannose-sensitive hemagglutinin (
msh) as well as reduction of flagellar gene expression in response to variability in environmental concentrations of the second messenger 3′, 5′-cyclic diguanylic acid (c-di-GMP) which aids in the integration of environmental stimuli and effects cell physiology for many microorganisms.
The genetic predisposition of
V. cholerae to transition between environmental and pathogenic lifestyles was recognized upon sequencing its two chromosomes (
Heidelberg etal., 2000). Results indicate that the small chromosome has a greater proportion of hypothetical genes (59%) than the large chromosome (42%) and has more genes originating in plasmids and other γ-Proteobacteria. In addition to providing evidence that the small chromosome originated as a mega-plasmid which was acquired by an ancestral
V. cholerae, this study provides a foundation for deciphering how these environmental bacteria emerged as enteric pathogens.
Additional factors contributing to successful transition at the environment-host interface were investigated by Vezzulli etal. (
2008) who identified dual role colonization factors (DRCF) such as the cholera toxin (
ctx), toxin co-regulated pili (
tcp), alternative sigma factor (
rpo) and quorum sensing genes (
lux) that are necessary for successful colonization of both the gastrointestinal epithelia and the chitinous exoskeleton of copepods in the aquatic environment. Further, Kirn etal. (
2005) recently recognized a gene (
gpbA) involved in chitin colonization in the aquatic environment that is also necessary for successful colonization of the epithelia in the mammalian host. Exoskeleton associations have been shown to be central for environmental survival, biofilm formation and intra-species gene exchange, as well as pathogenicity and pre-conditioning of ASR (
Nalin, 1976;
Nalin et al., 1979;
Blokesch and Schoolnik, 2007;
Pruzzo et al., 2008). The significance of these associations is further illustrated by the observation that Cholera epidemics are seasonal and strongly linked to algal blooms due to
V. cholerae associations with phytoplankton and the chitinous exoskeleton of copepods within aquatic systems (
Colwell, 1996;
Hsieh et al., 2007).
Conclusions
Since the discovery of acid stress response and its characterization in E. coli and S. typhimurium, there has been a broad array of highly conserved acid stress response factors observed in Gram-negative and Gram-positive pathogens with new model organisms being reported. The use of large-scale, signature-tagged mutagenesis (STM), microarrays and metatranscriptomic analysis have led to rapid discovery of novel factors for acid resistance and provide insight into the mechanisms of this bacterial stress response. However, the role of various environmental reservoirs on acid adaptation and elicitation of the ASR in the context of pathogen ecology remains largely unexplored. Studies have focused upon bacterial pathogen response either within the mammalian host, in laboratory conditions mimicking the host or in industrially relevant food products during food preservation and processing. While understanding of ASR is immensely valuable and central to the control of microbial pathogens, gaining a deeper understanding of environmental reservoirs that condition for acid adaptation and the elicitation of stress response pathways will enable disruption of the fecal-environmental-oral cycling of these enteric pathogens. Such insights would aid in developing reliable models of pathogen distribution that effectively incorporate the functional niche of microorganisms and the environmental ‘hot spots’ of pathogenicity. Finally, characterizing these stress response pathways in environmental and clinical strains of enteric bacteria will provide a broader and more complete context of pathogenicity which could facilitate improved methods for bacterial control during food and water preparation and potentially aid in disease mitigation.
Higher Education Press and Springer-Verlag Berlin Heidelberg
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