| The intraceHular pathogen Listeria monocytogenes (L. monocytogenes, LM) is a gram-positive foodborne pathogen that thrives in diverse environments such as water, soil and various food products. The disease caused by this bacterium, listeriosis, is acquired by ingesting contaminated foods and mainly afffects the old people, pregnant women, infants and immunocompromised individuals. Listeriosis is considered an important threat to human health because it can be associated with severe disease, such as septicaemia, central nervous system infections, gastroenteritis and mater-nal-foetal infections including abortion, stillbirth, preterm birth and neonatal listerio-sis, resulting in high mortality up to30%. From1980s some human listeriosis cases have been reported every year in the Europe and North America. According to statistic, an average of2.2confirmed outbreaks per year were reported in the United States during1998-2008. The largest listeriosis outbreak in U.S. history occurred in2011, when147illnesses,33deaths, and1miscarriage occurred among residents of28states; the outbreak was associated with consumption of cantaloupe from a single farm. In China, a total of147clinical cases,479Listeria isolates and82out-break-related cases were reported in28(90%) provinces from January1964to De-cember2010.L. monocytogenes enters into host cells and spreads from cell to cell has been investigated in great detail and each stage is sophisticatly controlled by specific viru-lence factors, including internalins, intracellular proliferation and migration factors, the PrfA and SigB regulators, the two component systems and virulence-related small RNAs. L. monocytogenes is well adapted to the conditions during in the infection process and pursue different strategies to conteract changes in acidity, osmolarity and oxygen tension. Acidic environments are the most commonly encounted stress by L. monocytogenes at a number of stages during the course of its infectious cycle, from food to the gastrointestinal tract and finally in the phagosome of macrophages. Unlike many other microbes, Listeria possesses a number of potential mechanisms for pH homeostasis and hence can survive and proliferate to high numbers in acidic condi-tions. The exploration to the acid tolerance mechansims of L. monocytogenes will help to simulate and further understand its growth and proliferation in contaminated foods, which thus is able to effectively prevent and finally control human listeriosis caused by the bacterium.We this study attempt to elucidate:(1) L. monocytogenes strains variation in re-sponse to acidic conditions and the different inhibitory effects of various organic and inorganic acids on this bacterium;(2) the acid tolerance functions and its molecular mechanisms of L. monocytogenes arginine deiminase (ADI) system in host gastroin-testinal environment and its relationship with bacterial virulence;(3) the differences in molecular mechansims of L. monocytogenes agmatine deiminase homologs AguAl and AguA2mediating acid tolerance;(4) the regulatory mechanisms of L. monocyto-genes arginine repressor (ArgR) involved in arginine metabolism and acid tolerance, and its cross-regulation relations with SigB.1. The intracellular pH (pHi) measurement method of L. monocytogenesHere in this research, we innovatively proposed a more simple and effective ap-proach by using the pH-sensitive fluorescent probe CFDA-SE to study the varying viabilities and acid sensitivity of LM strains (EGDe,10403S,850658and M7) under different acidic conditions through monitoring the dynamics of pHi changes. The cal-ibration curves (Ratio490/435versus pHi) were performed using ethanol-treated cells of L. monocytogenes under different acids including hydrochloric acid (HCl), acetic acid (AA), citric acid (CA) and lactic acid (LA) in BHI broth for each strain (EGDe,10403S,850658and M7). The Ratio490/435and pHi values in each case were found to be best fitted by a third degree polynomial curve with a correlation index of over0.95and the detection limit of pHi can reach around5.5, which can be applied for further pHi determination under various acidic conditions.2. Acid tolerance of L. monocytogenes strainsLM strains (EGDe,10403S,850658and M7) show a great deal of variation in their response to acidic conditions. In BHI broth pre-justed by pH4.5and5.5organic or inorganic acids, the growth ability of10403S was nearly equal to850658, slightly stronger than EGDe, however significantly better than M7. Under these conditions, all the pHi dynamic values of EGDe,10403S,850658rebounded rapidly after a sharp decline (in5min) and then maintained a steady state, but there was no upward trend of M7after sudden drop. The pHi value in the specific time point of M7was signifi-cantly lower than the other three strains under the same acidic condition. In the case of pH3.5, all the strains began to die (survival rate is less than100%) and M7showed the most sensitivity to acid stress compared to10403S, EGDe and850658. Further-more, the survival rates of M7,10403S, EGDe and850658were0.03%,1.63%,1.17%and2.71%, respectively after exposed to the synthetic human gastric fluid (pH2.5) for30min. To conclude based on these data, LM strains vary widely in their sen-sitivity to acidic conditions.According to the growth ability assay in BHI broth pre-justed by pH4.5and5.5organic or inorganic acids, we found the differences in bacterial inhibitory were ob-vious. All the strains (M7,10403S, EGDe and850658) showd the most sensitivity to AA followed by LA. The CA and HA showed the considerable degree of inhibitory effect. In the case of pH3.5, all the strains began to die and none of the survival bac-teria were detected when exposed to the AA for60min. The pHi values of all strains exposed to protonated molecules of AA and LA is lower than that of cells exposed to HC1and CA at the same external pH. Overall, the bacterial inhibition effect of organic acid was significantly stronger than that of HA all in different strains. Therefore, we confirmed the weak acid could be used as an important food preservative in our study, which can help to prevent the growth of L. monocytogenes and enhance optimal use of organic acid in food production and preservation.3. Molecular mechanisms of L. monocytogenes arginine deiminase involved in acid toleranceBioinformatic analysis indicates that L. monocytogenes arcA encodes a protein containing conserved motifs (FTRD, EGGD, MHLDT and CMSxP) and a catalytic triad (Cys-His-Glu/Asp) characteristic of arginine deiminase that mediates arginine deimination reaction. We attempted to characterize the role of ArcA in acid tolerance in vitro and in mice models. Transcription of arcA was significantly increased in L. monocytogenes culture subjected to acidic stress at pH4.8, as compared with that at pH7.0. Deletion of arcA significantly impaired growth of L. monocytogenes under mild acidic condition at pH5.5(P<0.01) but not at pH7.0. Compared to wild-type strain, the survival ability of arcA mutant in synthetic human gastric fluid at pH2.5was markedly reduced. Furthermore, the arcA deletion mutant exhibited significant lower survival in murine stomach compared with that of wild-type at1h (Logio CFU:2.9vs5.8, P<0.01) or2hs p.i.(Logio CFU:1.8vs4.0, P<0.01). Bacterial load num-bers recovered from spleens of mice intraperitoneally inoculated with arcA deletion mutant were significantly lower than with the wild-type strain48h (Logio CFU:4.1vs6.7, P<0.01) and72h p.i.(Log10CFU:3.8vs6.9, P,0.01). These phenotypic changes were recoverable by genetic complementation. The pHi value in the specific time point of the mutant was significantly lower than wild-type under the pH3.5acidic condition (P<0.01). However, ArcA might not play a part in listerial intracellu-lar replication within macrophage cells as there were no significant differences in the fold-increase of the bacteria in RAW264.7cells among the arcA mutant and its parent strain (P>0.05). Thus, we conclude that L. monocytogenes arcA not only mediates ac-id tolerance in vitro but also participates in gastric survival and virulence in mice.4. Differences in molecular mechansims of L. monocytogenes agmatine deimi-nase homologs AguA1and AguA2mediating acid toleranceThe food-borne pathogen L. monocytogenes has the ability to survive and adapt to the low pH environment and subsequently cross the intestinal barrier, to establish successful infection. L. monocytogenes genes aguA1and aguA2both encode two pu-tative agmatine deiminase (AgDI), respectively named as AguA1and AguA2, which catalyzes the conversion of agmatine to N-Carbamoyputrescine, ammonia and ATP. The predicted protein structures of AguAl and AguA2show strong homology to the typical AgDIs and possess five putative catalytic residues (Asp94, Glu155, His216, Glu218and Cys356). Both the transcription of aguA1and aguA2was induced ap-proximately13and15folds, respectively, higher at acidic pH5.0than at neutral pH7.0. In addition, absence of aguAl significantly impaired its survival ability in syn-thetic human gastric fluid (pH2.5)(P<0.01), but not with aguA2. Furthermore, we found that only the mutants ΔaguA1and ΔaguA1ΔaguA2showed significantly lower survival in mice stomach than the parent strain10403S (Logio CFU:5.7and5.5vs6.6, P<0.01). Complementation of aguAl restored its survival to a level (Log10CFU:6.5) similar to the parent strain. These findings suggest that only AguAl, but not AguA2, is involved in listerial resistance to acid stress, although both were induced at the mRNA level under the acidic condition. Recombinant agmatine deiminases were overexpressed in E. coli and purified to homogeneity in high yield (～20and～40mg/L culture for AguA1and AguA2, respectively). The purified AguA1had a high specific activity of2050.78U/mg and its kinetic characterization has also been determined. With agmatine as the sole substrate, the Km, Vmax, kcat and kcat/Km values of AguAl were0.646±0.227mM,85.69±7.58μM/min,34.28±3.03min-1and5.304×104min-1M-1, respectively. However, AguA2showed no deiminase activity whichever substrate was used. AguAl exhibited high agmatine deiminase activity in a wide range of pH conditions from3.5to10.5with optimal pH at7.5, suggesting that the activity of AguA1is not sensitive to pH, which is consistent with its contribution to acid tolerance under low pH conditions. However, AguAl showed varying activities at different temperatures, optimal at25℃. Metal ions Cu2+, Zn2+and Co2+inhibited AguAl with varying degrees of potency with respective IC50of0.034±0.008mM,0.25±0.05mM, and2.89±0.82mM, indicating that copper ion is the most inhibitory. Furthermore, We have confirmed experimentally that the mutants in AguA1(D94A, D94E, E155A, E155D, H216A, H216R, D218A, D218E and C356A) completely abolished the enzymatic activity, indicating that the key residues play irreplaceable roles in enzymatic catalysis and substrate binding. Interestingly, we have found that AguA2acquired the agmatine deiminase activity only when Cys157was mutated to Gly as the same in AguAl and in turn AguAl with G157C mutation lost the full en-zymatic activity, which indicated that Gly157is a newly discovered key residue other than the known ones critical for AgDI activity. We also proposed a catalytic mecha-nism of L. monocytogenes AgDI based on these studies, which provide a better model to futher elucidate the molecular mechanism of this protein family mediating the bac-terial acid tolerance. In conclusion, our study revealed that only AguAl but not AguA2possesses the AgDI activity and mediates acid tolerance in L. monocytogenes.5. Regulatory mechansims of L. moconcytogenes arginine repressor (ArgR) in-volved in acid toleranceL. monocytogenes arginine repressor (ArgR) belongs to the ArgR/AhrC family of transcriptional regulators, which are involved in the regulation of arginine metabolism. The predicted protein folding of L. monocytoegenes ArgR shows strong homology to the typical ArgRs having a hierarchical structure with the hexamer made up of two tripmers, and conservatively possesses two critical domains ("SR" and "GTIAGDTT"). The "SR" and "GTIAGDTT" motifs are known as a DNA and argi-nine binding doman, respectively. We this study found ArgR was closely relatively to acid tolerance of L. monocytogenes. Compared to wild-type strain, the survival ability of argR mutant in acidic BH1medium at pH2.5was significantly increased, but in-tracellular replication within macrophage RAW264.7cells was markedly reduced. The promoter regions of arcA, sigB, argC and argG containing the putative ArgR boxes were respectively incubated with ArgR in vitro for protein-DNA binding by EMSA and the shift migrations with varying degrees were found in different promot-ers (The binding capacity order:argC≥argG>sigB>arcA). We further found LmArgR abolished or obviously impaired the binding ability to ArgR box when S42R43were both mutated to A42A43, confirming that S42R43are critical amino acids for ArgR-DNA binding. Under pH5.5and pH7.0conditions, both the transcript and protein levels in ArgR mutant strain were upregulated with extremely significant de-gree. To further analyse the regulatory function of ArgR in arginine biosynthesis re-lated genes, we cloned the promoter region of argG into the gfp3reporter vector and transformed it into wild-type and ArgR mutant strains. We found the ArgR mutant strain was able to induce strong fluorescent activity both under pH5.5and pH7.0conditions. In contrast, nearly no gfp expression could be detected in the wild-type, which further concluded that ArgR also plays a classical role of ArgR/AhrC family in feedback inhibitory of arginine biosynthesis pathway. ArgR deletion resulted in a cer-tain degree of increased protein expression of SigB and ArcA under non-stressed con-dition. When exposed to the acid stress, both the transcript and protein levels of sigB and arcA were significantly upregulated. We speculated that increasing of the acidic survival in the absence of ArgR was due to the upregutation of arcA and sigB. How-ever, the transcript of arcA and sigB were derepressed when exogenous arginine was added, and the in-depth studies are needed to clarify the underlying regulatory mech-anisms.In conclusion, this study demonstrated that (1) L. monocytogenes have strains variation in response to acidic conditions and the different inhibitory effects of vari-ous organic and inorganic acids on this bacterium;(2) L. monocytogenes arcA not on-ly mediates acid tolerance in vitro but also participates in gastric survival and viru-lence in mice;(3) L. monocytogenes AguAl but not AguA2possesses the AgDI activ-ity and mediates acid tolerance. More importantly, we have discovered Gly157as a noval key residue other than the known catalytic triad (Cys-His-Glu/Asp) that is criti-cal for enzyme activity;(4) L. monocytogenes ArgR plays a classical role of ArgR/AhrC family in feedback inhibitory of arginine biosynthesis pathway, and regu-lates the transcript and expression of arcA and sigB both in the arginine dependent and independent manners. |
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