Comparative Genomics And Pathogenicity Difference Mechanisms Of Listeria Monocytogenes Lineage â…¢ Virulent And Low Virulent Strains

Listeria monocytogenes, a Gram-positive non-spore forming rod, is resistant to a number of environmental stresses and able to survive or even replication in wide pH range (4.5-9), temperature range (0-45℃) and high salt (10% NaCl). It is an important zoonotic foodborne pathogen and causes septicemia, meningitis, and abortion in pregnant women with mortality as high as 30%. L. monocytogenes is classified into four lineages. Majority of the foodborne illness were caused by lineages I and II strains. Although strains of lineages III and IV rarely cause human outbreaks, there is still risk because these strains are diverse in virulence with some being highly pathogenic.L. monocytogenes encodes a set of virulence factors involved in intracellular infection:adhesion and invasion into host cells via receptor-specific InlA and InlB; escape from phagocytic or internalized vacuoles by membrane-damaging LLO, PlcA and PlcB; intracellular multiplication; and cell to cell spread via ActA-induced host cell actin polymerization for the new infection cycle. These virulence factors are delicately controlled by PrfA. We have two lineage III strains of significant difference of pathogenicity in murine model as well as marked difference in expression of PrfA-regulated virulence genes. L. monocytogenes encodes multiple sets of acid tolerance systems that operate independently. Recent research has indicated that the glutamate decarboxylase system (GAD) has major contribution to acid tolerance. GAD is composed of three glutamate decarboxylases and two glutamate transporters coded for in discrete genome regions. However, there is paucity of information on individual roles of these GAD components in acid tolerance and the regulatory mechanisms in response to stresses. In this study, we attempted to (1) explore the molecular basis governing the difference of pathogenicity between two lineage III strains by comparative genomics; (2) investigate the roles of constitutively expressed PrfA and relevant virulence factors controlled by PrfA in determining the virulence difference between the two strains; and (3) elucidate the mechanisms of differences of survival under acidic conditions by different GAD components and their regulation.I. Comparative genomic analysis of Listeria monocytogenes lineage III strains of high and low pathogenicityThe strain M7 and Lm850658 are serovar 4a strains belonging to the lineage III and have similar biochemical patterns and composition of virulence genes. Their pathogenicity varies greatly. Although strain M7 has high lipolytic and hemolytic activities, Lm850658 was far more effective in adhesion, invasion and cell-to-cell spread and more pathogenic to mice than M7. Comparative genomic approaches were then attempted to decipher the genetic basis that might govern the strain-dependent pathotypes. The genome size of Lm850658 and M7was 2,918,502 bp and 2,976,163 bp respectively, coding for 2885 and 2977genes. The two strains share the 2,761 ORFs of identical nucleotide sequences. The specific genes were 33 for Lm850658 and 130 for M7. There is a cryptic 20K region that is clearly a prophage containing 23 ORFs specific in the virulent strain Lm850658. The strain M7 has its specific genes contained in two large prophages (38K and 44K) that encode 52 ORFs and 69 ORFs, respectively. The 20K prophage is the first one found in Listeria with no homologous sequences in the NCBI database. The 38K and 44K prophages are conserved in strains M7, HCC23 and L99. To examine the roles of these specific prophages in pathogenicity, the 20K and 38K prophages were deleted from their respective strains Lm850658 and M7 by homologous recombination. There were virtually no differences of pathogenicity shown as adhesion, invasion, cell-to-cell spread and in mouse model between the deletion mutants and their parent strains although some putative virulent factors like the VirB4 are present in the 20K region or holin-lysin in the 38K region. Therefore, the genes encoded in the 20K and 38K prophages of L. monocytogeness Lm850658 and M7 are not related to virulence.II. Effects of constitutively activated PrfA in Listeria monocytogenes lineage III strain of high and low virulence on pathogencity-related phenotypesThe amino acid mutations of L. monocytogenes PrfA (L140F, G145S, and G155S) lead to its constitutive expression. Sequence analysis shows that M7 is different from Lm850658 and EGDe by its PrfA145S, indicating constitutive expression in M7. The PrfA regulated genes in M7 had significantly higher expression than Lm850658 and EGDe in broth and whole blood. To uncover this contradictious relationship, we exchanged the prfA genes between strains Lm850658 and M7. For Lm850658, constitutive activation of PrfA potentiated its virulence-related traits shown as elevated hemolytic and lipolytic activity, increased adhesive and invasive abilities as well as enhanced cell-cell spread in cultured cell lines. Replacement with non-activated PrfA in M7 led to reduced adhesion and invasion as compared with its wild-type strain. Exchange of PrfA did not affect cell-to-cell spread and intracellular survival of both strains. However, PrfA activation did increase the pathogenicity of Lm850658 to immune compromised ICR mice. Western blotting of whole bacterial proteins with specific antibodies showed that some PrfA-regulated genes were over-expressed in vitro in constitutively activated PrfA strains. In contrast to virulent strain Lm850658, majority of InlB in M7 was detected in the culture supernatants and less on the bacterial surface while the reverse was seen in Lm850658. There was no significant difference in expression of ActA, LLO and InlA on the bacterial surface or in culture supernatants between the strains and/or the mutants.The above findings indicate that low virulence of strain M7 is due to its defects in infecting host cells as a result of failed anchorage on the bacterial cells of surface proteins like InlB, a major protein involved in adhesion and invasion of pathogenic L. monocytogenes strains. Although we did not see apparent difference of ActA distribution and abundance on the bacterial surface and in culture supernatants between the strains, there might also be abnormal anchorage on the bacterial surface that ultimately affect cell-to-cell spread and aggregation/precipitation in liquid medium of the M7 strain. Because InB and ActA are conserved between the two strains, and they bind to the cell wall by interaction with lipoteichoic acid via the GW motif, we postulate that abnormal anchorage of both proteins might be related to cell wall structure of M7. As such, further study should be directed to dissect the relationship between lipoteichoic acid metabolism and the anchoring proteins like InlB in an effort to decipher the mechanisms of M7 low virulence.Ⅲ. Comparison of components of Listeria monocytogenes glutamate decarboxylase system and their regulatory aspectsL. monocytogenes 10403S showed highest resistance to acidic stress, followed in order by Lm850658, M7 and EGDe. All strains have the FoFi-ATPase system. Strains 10403S and EGDe have complete sets of genes coding for arginine deminase (ADI), agmatine deminase (AgDI) and glutamate decarboxylase (GAD) systems. Strains Lm850658 and M7 lack genes coding for AgDI and gadDl/gadTl. With 10403S, gad2 contributes most to acid tolerance, followed in order by sigB, gadD3, augAl and arcA, while augA2 and gadDl are not involved in acid tolerance. The gadT2/gadD2 components exhibited highest transcription, followed by gadD3 and gadD1gadT1. The transcriptional levels of gadT2/gadD2 in strain 10403S were significantly higher that EGDe while those of gadDl/gadTl and gadD3 were similar between the two strains. Both M7 and Lm850658 had similar levels of gadD2 to 10403S, but their gadT2 transcription was low. Immunoblotting showed that all strains had similar levels of GadD2 expression except strain EGDe that did not show a visible band when treated and exposed under the same conditions. GadD2 expression was generally higher than GadD3. The results lead us to postulate that contribution to acid tolerance seems to be proportiaonal to expression levels of the components of the GAD system although they all play a role in such tolerance, and that expression of the GadT2/GadD2 component appears to be the major factor of strain-dependent difference in acid tolerance.Sequence analysis showed that the promoter regions of gadTl, gadT2 and gadD3 are highly conserved between strains 10403S and EGDe. The GFP reporter assay indicated that only PgadT2 and PgadD3 could drive gfp expression and their promoter activity differed among strains. PgadT2 was strong in 10403S and Lm850658, but weaker in M7 and not active in EGDe. PgadD3 had similar activity in 10403S and EGDe, but significantly lower than PgadT2 and no activity in Lm850658 and M7. These results suggest that the difference of acid tolerance among strains is dependent on the expression of GadT2/GadD2 which is regulated by certain transcriptional factors. By transcriptional analysis, we found that the GAD system components did not respond to acidic stimuli. Genes gadTl and gadT2/gadD2 were not regulated by SigB, a global stress response regulator, while gadD3 transcription seemed to be SigB-dependent. All components of the GAD system were not regulated by five putative GAD transcription regulators GadRs. Interestingly, a mild alkaline stress at pH 8.8 down-regulated gadT2/gadD2 transcription and PgadT2 activity. Therefore, the regulatory mechanisms and the responsible negative transcriptional regulators under alkaline stress that might govern differential expression of GadT2/GadD2 among strains of different strains require further investigation.In summary, this study clearly indicates that the three prophages in the lineage Ⅲ strains only contribute to genetic diversity, but not to pathogenicity. It is also clear that abnormal anchorage of virulence factors such as InlB onto the bacterial surface is the main factor of low pathogenicity of the lineage Ⅲ strain M7. We also provide clear evidence that the GadT2/GadD2 component within the GAD system and their expression level are the determine factors of acid tolerance in L. monocytogenes. These findings could facilitate further investigations into the pathogenesis and mechanisms of stress tolerance of L. monocytogenes.