Listeria Monocytogenes: Molecular Evolution And Functional Genomics Under Acidic Conditions

The intracellular pathogen Listeria monocytogenes is the causative agent of listeriosis, a severe invasive illness that has an extremely high mortality rate. This disease is primarily transmitted by consumption of contaminated foods. The incidence of listeriosis has increased in Europen and American countries.From 2007 through 2009, three large listeriosis outbreaks have been reported in US, Canada, and Denmark, which lead to 30 deaths and a large proportion of perinatal infections. Unfortunately, there is a paucity of systemic investigation on the prevalence of Listeria in Chinese food systems.L. monocytogenes encompasses a diversity of strains with varying virulence and pathogenicity. While serovar 4b strains caused the vast majority of invasive listeriosis cases with a higher mortality rate than other serovars, lineage III strains are rarely associated with human listeriosis. L. monocytogenes infection process comprises several distinct stages:tolerance to adverse conditions in gastrointestinal tract (GIT), adhesion and invasion of host cells, escape from vacuole, intracellular multiplication and intercellular spread. Each stage invovles specific virulence factors. After ingestion by host, L. monocytogenes encounters the stomach with low pH. Therefore, survival in acid environments is the primary ability for L. monocytogenes to initiate infection. L. innocua is most closely related to L. monocytogenes, and they usually co-exist in food specimens.Although these two species resemble each other ecologically, biochemically and genetically, L. innocua has no pathogenic inclination. Therefore, the L. monocytogenes-L. innocua clade within the genus Listeria can be used as a model system to examine the evolution of pathogenicity.This study was in an attempt to clarify (1)molecular epidemiology and pathogenic potential of L.monocytogenes isolates from Chinease food system and imported food products;(2) the biodiversity and evolution of L. monocytogenes-L. innocua clade;and (3)the role of arginine deiminase system in acid tolerance/resistence and its underlying molecular mechanisms.1.Molecular epidemiology and pathogenicity of L. monocytogenes in food systemsA novel multiplex PCR, based on Imo0038 in combination with optimized iap migration profiles, was developed for simultaneous identification of Listeria species and discrimination of L. monocytogenes lineage III. The recovery rate of Listeria in Chinese food products between 2000 and 2007 was 3.7%, with L. monocytogenes accounting for 25.3%.There was no difference between the recovery rate in summer and that in winter. Of the seven main categories of food products, meat and seafood represented the major products to be contaminated by Listeria. Among the 88 selected L. monocytogenes food isolates from Zhejiang and Fujian provinces, serovar 1/2a dominated (47.7%),and the isolation of serovar 4b (6.8%) and lineageⅢ(2.4%) was low. From the 1275 batches of aquatic products imported from 29 countries, the recovery rate of Listeria was 2.8%, similar to that in Chinese aquatic products (2.7%). However, L. monocytogenes accounted for a surprisingly high proportion (91.7%) compared to that in Chinese aquatic products (22.2%).Serovar 4b predominted (65.2%), with epidemic clonesⅠ(ECⅠ) andⅡ(ECⅡ) recognized, followed by serovar 1/2a(13.0%).In the cladogram, ECI formed sister branch with clinical isolates from Chinese diseased animals, while ECⅡwas placed between 1/2b isolates.Chinese food 4b isolates occupied another branch not related to any ECs. All L. monocytogenes isolated from Chinese food systems and imported aquatic products harbored LIPI-1 and InlAB (except S10), and demonstrated virulence potential in mouse model except M7 being low-pathogenic.The absence of one proline-rich repeat (PRR) did not stop the bacterium causing listeriosis, but offered a potential marker for differentation of epidemic clonesⅠandⅡfrom phylogenic perspective. LineageⅢwas recovered at an extremely low frequency, but might serve as sources of clues for understanding the evolution history in L. monocytogenes-L. innocua clade.2. Diversity and evolution of L. monocytogenes lineageⅢIn the cladogram based on 16S rRNA,13 L. monocytogenes lineageⅢstrains were placed between L. monocytogenes, L. innocua and a novel species, L. marthii. The phylogenetic tree based on 21 genes revealed three major branches representing sublineagesⅢA,ⅢB andⅢC respectively.ⅢA was further separated intoⅢA-1,ⅢA-2 andⅢA-3(containing low-pathogenic strains M7 and 54006).These lineageⅢstrains were grouped into 8 biochemical types (BTs) based on 46 biochemical reactions.ⅢA-3 belonged to BT1,ⅢA-1 andⅢA-2 to BT2,ⅢB to BT3 to BT7 (5ⅢB strains representing 5 individual BT), andⅢC to BT8. These lineageⅢstrains were separated into 10 internalin type (ITs)dusted in 4 categories.ⅢB belonged to categoryⅠcontaining the least number of internalins,ⅢA-3 andⅢC to categoriesⅡandⅢrespectively, andⅢA-1 andⅢA-2 to categoryⅣwith the most number of internalins.The organization of internalin genes between ascB and dapE further classifiedⅢB into andⅢB-1 andⅢB-2, andⅢC intoⅢC-1 andⅢC-2.AllⅢA-1,ⅢA-2,ⅢB andⅢC strains showed comparable survival ability in human gastric fluid, adhesion and invasion of epithelial cells, intracellular spread ability and pathogenicity in normal and immunocompromised mice to those of lineagesⅠandⅡstrains.Thus, the uncommon human listeriosis cases due to lineageⅢstrains might be explained by the rarity of foodborne exposure to lineageⅢ.ⅢA-3 had an imparied survival ability in human gastric fluid and formed no visible plaques in fibroblast in the presnce of gentamycin. Also,ⅢA-3 was eliminated by host more easily than pathogenic strains, and thus exhibited negligible virulence in normal and immunocompromised mice.ⅢA-3 contained the complete version of LIPI-1 and InlAB.Lacking some internalins (e.g. InlC, InlF and InlJ) and one PRR in ActA did not contribute to the subdued virulence. PrfA is the main virulence regulator in L. monocytogenes, and the core PrfA regulon encompasses LIPI-1 and inlAB. The expression levels of LIPI-1 and InlAB inⅢA-3 were suprisingly higher than pathogenic strains, and declined drastically in the PrfA mutant inserted by transposon. Significant mutation of PrfA inⅢA-3 was found at 145 (Glycine to Serine), which introduced a repositioning of the PrfA helix-turn-helix DNA binding region in comparison to the wild-type structure. This repositioning resulted in the constitutive activation of PrfA and the overexpression of PrfA-regulated factors (e.g. LLO, PC-PLC), which faciliated the membrance-lysis process. Hence, bacteria was exposed to the immune system and easily cleared, which resulted in the reduced virulence.The complete genome ofⅢA-3 strain M7 was sequenced by Illumina/Solexa. M7 contained one circular chromosome of 2852640bp in length with an average G+C content of 38.2%, higher than those in other lineages.There were 2970 predicted coding regions in the genome. Compared to pathogenic strains, M7 lacked 109 genes clustered in 46 loci, including ADI island, rplS-infC internalin island, ascB-dapE internalin island,11 transcriptional regulators, etc, and possessed 345 specific genes, covering those encoding virulence-associated E family protein (M7-28), Sigma family protein (M7-210),internalin (M7-214),4 transcriptional regulators, etc.ⅢA-3 harbored LIPI-1 like pathogenic strains, and had higher nucleotide identity of 23S rRNA to L. innocua than to L. monocytogenes. In the cladogram based on 2168 genes conserved in genus Listeria,ⅢA-3 were placed between L. monocytogenes lineagesⅠandⅡand L. innocua. By bearing L. monocytogenes-specific virulence genes (e.g. LIPI-1 and inlAB), and sharing similar gene deletions (e.g.ADI island, rplS-infC and ascB-dapE internalin islands) and gene insertions (e.g.108 genes existing inⅢA-3 and L. innocua) with L. innocua, L. monocytogenesⅢA-3 constituted a evolutionary intermediate between L. monocytogenes and L. innocua. In addition,ⅢA-3 possessed many specific proteins involved in horizontal gene transfer (HGT) events, e.g. phage-related proteins, site-specific recombinases, integrases, conjugative transposon proteins and CRISPR-associated proteins, suggesting a higher frequency of HGT than that in other lineages. However, IIIA-3 failed to indicate the evolutionary direction of L. monocytogenes-L. innocua clade. As the other side of the coin, L. innocua might hold the key to clarifying the evolution direction.3.Population structure of L. innocua and evolution history of the L. monocytogenes-L. innocua cladeBased upon internalin profiling and multilocus sequence typing, L. innocua was separated into four subgroups.Subgroups A and B correlated with internalin types 1 and 3 (except strain 0063 belonging to subgroup C) and internalin types 2 and 4 respectively. The majority of L. innocua strains belonged to these two subgroups. The time to the most recent common ancestor (TMRCA) of L. innocua subgroups A and B were similar, suggesting these two subgroups appeared at approximately the same time. However, subgroup A harbored a whole set of L. monocytogenes-L. innocua common and L. innocua-specific internalin genes, and displayed strikingly higher recombination rates than those of subgroup B,including the relative frequency of occurrence of recombination versus mutation (ρ/θ) and the relative effect of recombination versus point mutation (r/m).Subgroup A also exhibited a significantly smaller exterior/interior branch length ratio than expected under the coalescent model, suggesting a recent expansion of its population size. Therefore, subgeoup A might represent the possible evolutionary direction towards adaptation to enviroments. All L. innocua strains lacked 17 virulence genes found in L. monocytogenes (except for the subgroup D strain L43 harboring inlJ) and were nonpathogenic to mice. L. innocua was genetically monophyletic compared to L. monocytogenes, representing a young species descending from L. monocytogenes. The evolutionary history in the L. monocytogenes-L. innocua clade represents a rare example of evolution towards reduced virulence of pathogens.Subgroup D, which correlated with internalin type 5,branched off from the other three subgroups, and served as another evolutionary linkage between L. monocytogenes and L. innocua. This subgroup could be differentiated from other subgroups by Asp-Phe-Pro arylamidase and alanine arylamidase reactions.Compared to subgroup A strain CLIP11262, subgroup D lacked 365 genes clustered in 50 loci, some of which were also absent in L. monocytogenes or L. monocytogenes IIIA-3. Subgroup D contained L. monocytogenes-specific virulece gene inlJ. However, this gene carried a premature stop codon mutation that led to production of a truncated and possibly nonfunctional InlJ due to a non-in-frame deletion of seven nucleotide acids within the 11th PRR. Thus, subgroup D failed to invade and spread efficiently.Overall, a stepwise evolution model of L. monocytogenes-L. innocua clade was demonstrated. If we consider gene deletion as an important force in Listeria evolution, L. monocytogenes serovar 1/2c (lineageⅡ) is more ancestral in this clade, and evolved in three directions. One evolved into serovar 4b (lineageⅠ) via serovar 1/2b (lineageⅠ); one gave rise toⅢB (fromⅢB-1 toⅢB-2);and the other turned into serovar 1/2a (lineageⅡ) andⅢA-1/2, and the latter further intoⅢC (fromⅢC-1 toⅢC-2) andⅡA-3.ⅢA-3 evolved into L. innocua via subgroup D.4. Functional genomics of L. monocytogenes under acidic conditionsBased upon the comparison of gene transcription profiling at neutral (pH7.0) and acidic (pH4.8) conditions using Solexa genome analysis system, two systems were identified from the whole genome of 10403S with elevated transcriptional levels under acidic conditions.One was the glutamate decarboxylase (GAD) system, and the other was the putative arginine deiminase (ADI) system (lmo0036-lmo0043 cluster). The GAD system involved three GAD paralogs (gadDl,gadD2, gadD3) located in three distinct loci.The transcription of gadD2 and gadD3 were increased under acidic condition, indicating a role of GAD system in acid tolerance. Moreover, it is suggested that GAD system co-evolved with inlGHE and inlGC2DE.From perspectives on molecular evolution and functional genomics, ADI gene island is of great interest to us.This island comprised eight genes, arc family genes arcABCD, aguA family genes aguAl and aguA2, one putative transcriptional regulator lmo0041,and one unknown gene lmo0042, which involved in arginine deiminase and agmatine deiminase systems.The transcription of those genes were increased under acidic condition relative to those under neutral condition. The AarcA,ΔarcB,ΔarcD,ΔaguA1 andΔaguA2 null mutants exhibited impaired growth rate under low pH (pH 5.5).Deletion of these genes also led to a decreased survival rates in synthetic human gastric fluid (pH 2.5).In addition, these knock-out mutants demonstrated a lower pathogenicity to mice relative to wild-type strain. Remarkably, aguAl and aguA2 represented two agmatine deiminase (AgDI) paralogs, but AguA2 contributed to acid tolerance and pathogenicity more effectively.5. Molecular mechanisms of the arginine deiminase-agmatine deiminase system in L. monocytogenes arcA (lmo0043) encoded ADI using arginine as substrate. L. monocytogenes harbored the whole arg gene family, suggesting arginine could be synthesized de novo. Also, L.monocytogenes was able to uptake extracellular arginine under strong acid conditions.ADI triggered the ADI pathway, and mediated the first reaction producing citrullin and ammonia. aguAl (lmo0038) and aguA2 (lmo0040) encoded two AgDI paralogs with agmatine as substrate.L. monocytogenes was able to produce agmatine from arginine using arginine decarboxylase, and uptake extracellular agmatine under strong acid conditions. AgDI initiated the AgDI pathway, and mediated the first reaction producing carbamoylputrescine and ammonia. Interestingly, although AguAl and AguA2 showed similar hydrophilicity and contained similar active sites, AguA2 played a more important role in acid tolerance.ArcB (lmo0036) displayed the ornithine carbamoyltransferase (OTC) and putrescine carbamoyltransferase (PTC) activities, representing the first example of carbamoyltransferase responsible for two sets of reversible reactions,in vitro kinetic studies showed that the equilibrium of the reaction lied overwhelmingly towards the formation of citrulline or carbamoylputrescine (anabolic reaction).While the optimum pH for anabolic reactions were at pH 8 to pH 10, the optimum pH for catabolic reactions were at pH 5 to pH 5.5,indicating ArcB possibly served as catabolic OTC or PTC under acidic conditions. While catabolic OTC mediated the reaction yielding ornitine and carbamoyl phosphate, catabolic PTC mediated reaction with the products of putrescine and carbamoyl phosphate. Thus, ArcB linked the second and following reaction steps, and promoted the ADI and AgDI pathways.Carbamoyl phosphate turned into ammonia, carbon dioxide and ATP by carbamate kinase (CK).Ornithine or putrescine was transported out of the cell in exchange for a molecule of arginine or agmatine by a membrane-bound antiport (AP) encoded by arcD (lmo0037), initiating a new metabolic circle at a state of dynamic equilibrium. For each mole of arginine catabolized via the ADI or AgDI pathway, two moles of ammonia were produced. This ammonia combined with intracellular cytoplasmic protons to yield ammonium inos (NH4+), thereby alleviating acidification of the cytoplasm and maintaining pH homeostasis.Beneath the exquisite organization of ADI system, it revealed a rigorous transcriptional network. Lmo0041,a rpiR family transcriptional regulator, and ArgR, an arginine biosynthetic repressor, regulated ADI/AgDI pathways negatively and positively in the synthetic human gastric fluid. The repressive function of Lmo0041 took precedence over the activation of the ADI system mediated by ArgR. ArgR also elevated the expression of Lmo0041,which further facilitated its repression of ADI system. PrfA and stress regulator SigB might be at the top of the hierarchy of regulatory network, regulating ADI system as well as ArgR and Lmo0041,evidenced by the potential PrfA and SigB binding sites proceeding the genes in ADI gene island as well as argR.These regulators activated or repressed the ADI system under varied conditions, which mediated listerial acid tolerance and pathogenicity.In conclusion, this study demonstrated that(1)L. monocytogenes food isolates (except lineageⅢisolate M7) all exhibit virulence potential, among which the isolates from imported aquatic products pose higher risk than those from Chinese food system; (2) L. monocytogenes lineageⅢencompasses a diversity of sublineages, with sublineage IIIA-3 showing low virulence which possibly caused by G145S mutation in PrfA; (3)L. innocua is a young species, and comprises four subgroups, with subgroup A representing the possible evolutionary direction towards adaptation to enviroments, and subgroup D together with L. monocytogenes sublineageⅢA-3 serve as evolutionary intermediates of the L. monocytogenes-L. innocua clade, representing a rare example of evolution towards reduced virulence of pathogens;(4) GAD and ADI systems contribute to the acid stress responses in L. monocytogenes, and ADI system is also involved in the listerial pathogenesis; (5) ADI pathway and AgDI pathway are mediated by functional proteins, including ADI, two paralogs of AgDI, OTC/PTC and AP;and (6) ADI system is under the regulatory network.