Presentation of Listeria monocytogenes antigens by major histocompatibility complex class I molecules to CD8 cytotoxic T lymphocytes independent of listeriolysin secretion and virulence

Virulence and intracellular persistence of Listeria monocytogenes markedly depend on secretion of listeriolysin (Hly), which promotes invasion of the pathogen from the endosome into the cytosol. Recent studies have provided compelling evidence that Hly also facilitates recognition of listerial antigens, in association with major histocompatibility complex (MHC) class I molecules, by CD8 T lymphocytes. Data presented here confirm that the Hly-deficient strains, the prfA^? mutant L. monocytogenes SLCC53 and the transposon mutants L. monocytogenes M3 and M20 are avirulent for mice, and unable to replicate inside bone marrow-derived macrophages (BMMΦ). Furthermore, BMMΦ infected with M3, M20 or SLCC53 were as efficiently lysed as BMMΦ infected with the Hly-positive wild-type strain EGD by MHC class I-dependent CD8 cytotoxic T lymphocytes. Using the highly sensitive polymerase chain reaction method, hly mRNA was detectable in BMMΦ infected with L. monocytogenes EGD or SLCC53, but totally absent in M3-infected BMMΦ. In the case of M20, an excision of the transposon occurred, but the excision was not precise and the hly gene was approximately 400 base pairs shorter. These findings argue against a unique role for Hly in MHC class I presentation of listerial antigens, although Hly appears central to virulence and intracellular replication. Thus, virulence of L. monocytogenes is dissociable from MHC class I presentation of listerial antigens.     

Sequence heterogeneity of PsaA, a 37-kilodalton putative adhesin essential for virulence of Streptococcus pneumoniae.

psaA encodes a 37-kDa putative pneumococcal surface adhesin. Although its complete nucleotide sequence has been determined, its contribution to the pathogenicity of Streptococcus pneumoniae has not previously been assessed. In this study, we used a PCR-amplified internal fragment of the psaA gene from S. pneumoniae type 2 strain D39 cloned in pVA891, to direct the construction of D39 derivatives in which the psaA gene had been specifically interrupted, by insertion-duplication mutagenesis. Two independent D39 psaA mutants (PsaA-(1) and PsaA-(2)) were significantly less virulent (as judged by intranasal or intraperitoneal challenge of mice) than either the wild-type D39 strain or a derivative of PsaA-(1) in which the psaA gene had been reconstituted by back-transformation with an intact copy of the cloned gene. pVA891-directed mutagenesis of an open reading frame (designated ORF3) immediately 3' to psaA or insertion of pVA891 between psaA and ORF3 had no impact on intranasal virulence. However, a small but significant difference in virulence was observed between these two derivatives and the parental D39 strain in a low-dose intraperitoneal challenge model, suggesting that the ORF3 product may also contribute to pathogenesis. Adherence of PsaA-(1) to A549 cells (type II pneumocytes) was only 9% of that for D39, while the ORF3-negative strain exhibited intermediate adherence (23%). This is the first functional evidence that PsaA is an adhesin. Sequence analysis of the psaA gene from D39 indicated significant deviation from that previously published for the homolog from S. pneumoniae R36A. The deduced amino acid sequences of mature PsaA from the two strains had only 81% homology, with the bulk of the variation occurring in the amino-terminal portion.     

The stress-responsive dgk gene from Streptococcus mutans encodes a putative undecaprenol kinase activity

We analyzed a previously constructed stress-sensitive Streptococcus mutans mutant Tn-1 strain resulting from disruption by transposon Tn916 of a gene encoding a protein exhibiting amino acid sequence similarity to the Escherichia coli diacylglycerol kinase. It was confirmed that the mutation led to significantly reduced lipid kinase activity, while expression of the intact gene on a plasmid restored both kinase activity and the wild-type phenotype. Further analysis revealed that the product of the dgk gene in S. mutans predominantly recognizes a lipid substrate other than diacylglycerol, most likely undecaprenol, as demonstrated by its efficient phosphorylation and the resistance of the product of the reaction to saponification. The physiological role of the product of the dgk gene as a putative undecaprenol kinase was further supported by a significantly higher sensitivity of the mutant to bacitracin compared with that of the parental strain.     

Novel role of the lipopolysaccharide O1 side chain in ferric siderophore transport and virulence of Vibrio anguillarum

From a library of approximately 20,000 transposon mutants, we have identified mutants affected in chromosomal genes involved in synthesis of the siderophore anguibactin, as well as in ferric anguibactin utilization. Genetic and sequence analyses of one such transport-defective mutant revealed that the transposon insertion occurred in an open reading frame (ORF) with homology to rmlC, a dTDP-rhamnose biosynthetic gene. This ORF resides within a cluster of four ORFs, all of which are predicted to function in the biosynthesis of this O side chain precursor. The same phenotype was seen in a mutant obtained by allelic exchange in rmlD, another ORF in this dTDP-rhamnose biosynthetic cluster. This mutation could be complemented with the wild-type rmlD gene, restoring both production of the O1 antigen side chain and ferric anguibactin transport. Presence of the O1 side chain was crucial for the resistance of Vibrio anguillarum to the bactericidal action of nonimmune serum from the fish host. Surprisingly, further analysis demonstrated that these mutations were pleiotropic, leading to a dramatic decrease in the levels of FatA, the outer membrane protein receptor for ferric anguibactin transport, and a concomitant reduction in iron transport. Thus, our results in this work demonstrate that the lipopolysaccharide O1 side chain is required for the operation of two critical virulence factors in V. anguillarum: serum resistance and anguibactin-mediated iron transport. These factors allow V. anguillarum to survive in serum and multiply in the iron-limiting milieu of the host vertebrate.     

Isolation and characterization of the Treponema denticola prtA gene coding for chymotrypsinlike protease activity and detection of a closely linked gene encoding PZ-PLGPA-hydrolyzing activity.

The chymotrypsinlike protease gene (prtA) from Treponema denticola ATCC 35405 was isolated from a lambda gt11 clone bank as one of several clones expressing protease activity. The DNA from one positive clone capable of hydrolyzing type IV collagen was subcloned into plasmid vector pUC119 for further analysis. Deletion analysis of subclone pXQ27.2 revealed the approximate location of the prtA gene on the DNA insert. DEAE-Sephadex chromatography of crude cell extracts of the subclone revealed two distinct T. denticola enzymes, one hydrolyzing SAAPNA (succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine-p-nitroanilide [chymotrypsin substrate]) and the other hydrolyzing PZ-PLGPA (phenylazobenzyl-oxycarbonyl-L-leucylglycyl-L-prolyl-D -arginine [collagenase substrate]). Each activity was purified to near homogeneity and exhibited by sodium dodecyl sulfate-polyacrylamide gel electrophoresis estimated molecular sizes of 67 and 36 kDa, respectively. Western blot (immunoblot) analysis demonstrated that only the 67-kDa SAAPNA-hydrolyzing enzyme reacted with antibody against the T. denticola chymotrypsinlike protease. The purified SAAPNA-hydrolyzing enzyme degraded type IV collagen, laminin, and fibronectin, but not type I collagen. These results indicate that the prtA gene coding for the chymotrypsinlike protease from T. denticola has been isolated. Another distinct gene encoding an enzyme hydrolyzing PZ-PLGPA appears to be adjacent to the prtA gene.     

Legionella dumoffii DjlA, a member of the DnaJ family, is required for intracellular growth

Legionella dumoffii is one of the common causes of Legionnaires' disease and is capable of replicating in macrophages. To understand the mechanism of survival within macrophages, transposon mutagenesis was employed to isolate the genes necessary for intracellular growth. We identified four defective mutants after screening 790 transposon insertion mutants. Two transposon insertions were in genes homologous to icmB or dotC, within dot/icm loci, required for intracellular multiplication of L. pneumophila. The third was in a gene whose product is homologous to the 17-kDa antigen forming part of the VirB/VirD4 type IV secretion system of Bartonella henselae. The fourth was in the djlA (for "dnaj-like A") gene. DjlA is a member of the DnaJ/Hsp40 family. Transcomplementation of the djlA mutant restored the parental phenotype in J774 macrophages, A549 human alveolar epithelial cells, and the amoeba Acanthamoeba culbertsoni. Using confocal laser-scanning microscopy and transmission electron microscopy, we revealed that in contrast to the wild-type strain, L. dumoffii djlA mutant-containing phagosomes were unable to inhibit phagosome-lysosome fusion. Transmission electron microscopy also showed that in contrast to the virulent parental strain, the djlA mutant was not able to recruit host cell rough endoplasmic reticulum. Furthermore, the stationary-phase L. dumoffii djlA mutants were more susceptible to H2O2, high osmolarity, high temperature, and low pH than was their parental strain. These results indicate that DjlA is required for intracellular growth and organelle trafficking, as well as bacterial resistance to environmental stress. This is the first report demonstrating that a single DjlA-deficient mutant exhibits a distinct phenotype.     

Salmonella pathogenicity island 2 influences both systemic salmonellosis and Salmonella-induced enteritis in calves

We have used signature-tagged mutagenesis to identify mutants of the host-specific Salmonella enterica serotype Dublin which were avirulent in calves and/or BALB/c mice. A mutant with a transposon insertion in the sseD gene of Salmonella pathogenicity island 2 (SPI-2), which encodes a putative secreted effector protein, was identified. This mutant was recovered from the bovine host but not from the murine host following infection with a pool of serotype Dublin mutants. However, a pure inoculum of the sseD mutant was subsequently shown to be attenuated in calves following infection either by the intravenous route or by the oral route. The sseD mutant was fully invasive for bovine intestinal mucosa but was subsequently unable to proliferate to the same numbers as the parental strain in vivo. Both the sseD mutant and a second SPI-2 mutant, with a transposon insertion in the ssaT gene, induced significantly weaker secretory and inflammatory responses in bovine ligated ileal loops than did the parental strain. These results demonstrate that SPI-2 is required by serotype Dublin for the induction of both systemic and enteric salmonellosis in calves.     

Characterization of a sialyltransferase-deficient mutant ofNeisseria gonorrhoeaestrain F62: instability of transposon Tn1545 Δ3 in gonococci and evidence that multiple genetic loci are essential for lipooligosaccharide sialylation

Neisseria gonorrhoeaestrain JB1 was previously shown to be defective in the sialylation of lipoologosaccharide (LOS) by exogenous CMP-NANA. The LOS components synthesized by the mutant now have been shown by mass spectrometry to be similar to those in the parental strain, F62, and to include the 4.5 kDa widely conserved lacto-N-neotetraose component that can be sialylated. The same two LOS components could be sialylated on the surface of the mutant and parental strains. One major component was sialylatable after chemical extraction of the LOS from either strain. These data confirm that the mutant, JB1, retains the ability to synthesize the LOS target required for the conversion by sialylation of serum-sensitive gonococci to serum resistance. A single base frame-shift mutation was found in thelstgene from the mutant, resulting in the replacement of the final 61 amino acids at the C-terminus of the sialyltransferase by four residues. Seventeen independent clones of thelstgene were isolated from the parental strain, but none of them complemented the sialyltransferase defect of the mutant and no sialyltransferase activity expressed from the clones could be detected inEscherichia coli.Although the data suggest that the mutant might be defective in genes at more than one chromosomal locus and that multiple loci are essential for sialyltransferase synthesis and activity, the alternative possibility, that DNA adjacent to thelstgene encodes a product which is toxic toE. coli, cannot be excluded. The site of insertion of the transposon Tn1545-Δ3 in strain JB1 was cloned and sequenced. The transposon is located in an intergenic region adjacent to genes for a putative ATP-dependent transport protein, but encoding no recognizable function relevant to LOS sialylation. Evidence that transposon Tn1545-Δ3 is unstable in gonococci is presented.     

Presence of a capsule in Erysipelothrix rhusiopathiae and its relationship to virulence for mice.

Three avirulent insertional mutants of Erysipelothrix rhusiopathiae were obtained by the technique of transposon mutagenesis with the self-conjugative transposon Tn916. The interactions between murine polymorphonuclear leukocytes and parent and mutant strains were studied in vitro. In the presence of normal serum, the virulent parent strain was resistant to phagocytosis, whereas the avirulent mutant strains were efficiently phagocytosed. In the presence of immune serum, the parent and the mutant strains were both efficiently phagocytosed. Electron microscopic examination of the parent strain demonstrated the presence of a structure resembling a capsule which was absent on the mutant strains, suggesting that a capsule may be involved in virulence. This was confirmed in studies in which an avirulent mutant strain reverted to virulence following acquisition of a capsule when the transposon was lost by spontaneous excision. These results strongly suggest that virulence of E. rhusiopathiae is associated, at least in part, with resistance to phagocytosis by polymorphonuclear leukocytes and that this antiphagocytic ability of the bacterium results from its possession of a capsule.     

Characterization of a ComE3 homologue essential for DNA transformation in Helicobacter pylori

To find genes involved in natural competence in Helicobacter pylori, we used a bioinformatics database search and found two transformation-related open reading frames (ORFs): a comE3 homologue (HP1361 ORF) of Bacillus subtilis and a comL homologue (HP1378 ORF) of Neisseria gonorrhoeae. We failed to obtain an HP1378 ORF knockout mutant, while an HP1361 ORF knockout mutant was obtained by transposon shuttle mutagenesis. The DNA transformation abilities of both natural transformation and electroporation were severely impaired (frequency, <10(-9)) in the HP1361(-) mutant. Complementation with a pHel2 vector carrying the HP1361 ORF restored the capabilities of natural competence (to a frequency of 4.21 x 10(-7)) and electroporation (to 3.62 x 10(-7)). The HP1361(-) mutant showed impairment in DNA binding and uptake. The results suggest that HP1361 is a comE3 homologue and is required for DNA binding and uptake during DNA transformation.     

Isolation and characterization of a capsule-deficient mutant of Actinobacillus pleuropneumoniae serotype 1

The capsular polysaccharides (CPS) play a major role in pathogenicity of Actinobacillus pleuroIpneumoniae, the causative agent of porcine pleuropneumonia. The purpose of the present study was to isolate a mutant in CPS biosynthesis by using a mini-Tn 10 transposon mutagenesis system and evaluate its adherence to host cells. One mutant apparently did not possess CPS as it did not react with a monoclonal antibody against A. pleuropneumoniae serotype 1 capsular antigen. Absence of capsule was confirmed by flow cytometry and also by transmission electron microscopy after polycationic ferritin labelling. The site of insertion of the mini-Tn 10 was determined and found to be in the cpxC gene. Its gene product, CpxC, is a protein involved in polysaccharide transport across the cytoplasmic membrane during CPS biosynthesis. Use of piglet tracheal frozen sections indicated that the CPS mutant adhered significantly (P=0.0001) more than the parent strain. The non-capsular mutant was less virulent in pigs compared to the parent strain and showed no mortality in experimentally infected pigs. The CPS mutant was however resistant to pig serum. This CPS mutant is the first A. pleuropneumoniae mutant in a CPS transport gene. It is also the first time that adherence of a CPS mutant of A. pleuropneumoniae is evaluated. Our observations indicate that capsular polysaccharides of A. pleuropneumoniae serotype 1 are not involved in adherence to piglet tracheal frozen sections but rather mask, at least in part, the adhesive functions.     

Galactose residues on the lipooligosaccharide of Moraxella catarrhalis 26404 form the epitope recognized by the bactericidal antiserum from conjugate vaccination

Lipooligosaccharide (LOS) from Moraxella catarrhalis has the potential to elicit bactericidal antibodies against the pathogen. We generated LOS-based conjugate vaccines that elicited bactericidal antibodies in animal models. However, epitopes on the LOS recognized by the functional anti-LOS antibodies remain unidentified. In this study, a mutant strain, D4, which lost the recognition by a bactericidal anti-LOS rabbit serum in Western blotting was generated from a serotype C strain 26404 by random transposon mutagenesis. Sequence analysis revealed there was an insertion of a kanamycin resistance gene in the lgt2 gene of D4, which encodes beta(1-4)-galactosyltransferase. An isogenic lgt2 mutant, 26404lgt2, was constructed. Structural analysis indicated that the mutant strain produced a truncated LOS lacking terminal galactoses from 4- and 6-linked oligosaccharide chains of strain 26404. Further studies showed that the antiserum lost the recognition of both mutant cells and LOSs in Western blotting, an enzyme-linked immunosorbent assay (ELISA), or a flow cytometry assay. The antiserum also lost the ability to kill the mutant strain in a bactericidal assay, whereas it showed a bactericidal titer of 1:80 to strain 26404. In an inhibition ELISA, d-(+)-galactose or 26404lgt2 LOS showed no inhibition. However, the 26404 LOS and a serotype A O35E LOS with terminal galactoses on its 6-linked oligosaccharide chain showed >90% inhibition, while a serotype B 26397 LOS showed >60% inhibition. These studies suggest that the terminal alpha-Gal-(1-->4)-beta-Gal on the 6-linked oligosaccharide chain of 26404 LOS plays a critical role in forming the epitope recognized by the bactericidal antiserum induced by immunization with our conjugate vaccine.     

Identification of a genetic locus essential for capsule sialylation in type III group B streptococci.

The type III capsular polysaccharide of group B streptococci (GBS) consists of a linear backbone with short side chains ending in residues of N-acetylneuraminic acid, or sialic acid. The presence of sialic acid on the surface of the organism inhibits activation of the alternative pathway of complement and is thought to be an important element in the virulence function of the capsule. We showed previously that a mutant strain of GBS that expressed a sialic acid-deficient, or asialo, form of the type III polysaccharide was avirulent, supporting a virulence function for capsular sialic acid. We now report the derivation of an asialo capsule mutant from a highly encapsulated wild-type strain of type III GBS, strain COH1, by insertional mutagenesis with transposon Tn916 delta E. In contrast to the wild-type strain, the asialo mutant strain COH1-11 was sensitive to phagocytic killing by human leukocytes in vitro and was relatively avirulent in a neonatal rat model of GBS infection. The asialo mutant accumulated free intracellular sialic acid, suggesting a defect subsequent to sialic acid synthesis in the biosynthetic pathway leading to capsule sialylation. The specific biosynthetic defect in mutant strain COH1-11 was found to be in the activation of free sialic acid to CMP-sialic acid: CMP-sialic acid synthetase activity was present in the wild-type strain COH1 but was not detected in the asialo mutant strain COH1-11. One of the two transposon insertions in the asialo mutant COH1-11 mapped to the same chromosomal location as one of the two Tn916 insertions in the previously reported asialo mutant COH31-21, identifying this site as a genetic locus necessary for expression of CMP-sialic acid synthetase activity. These studies demonstrate that the enzymatic synthesis of CMP-sialic acid by GBS is an essential step in sialylation of the type III capsular polysaccharide.     

Cloning and sequence analysis of the pfl gene encoding pyruvate formate-lyase from Streptococcus mutans.

We have isolated a sorbitol-negative mutant of Streptococcus mutans GS-5 following random mutagenesis with plasmid pVA891 clone banks. This mutant did not metabolize sorbitol anaerobically but did so aerobically. A 10-kb chromosomal DNA fragment flanking the pVA891 insertion was deleted in this mutant. The corresponding region from the parental strain GS-5 was then recovered by a marker rescue method with Escherichia coli. The pyruvate formate-lyase gene, pfl, was identified within a 3-kb PstI-XbaI fragment located in the middle of the deleted region of the chromosome, and its inactivation in S. mutans produced the same sorbitol-negative phenotype. Nucleotide sequence analysis of the pfl gene revealed a 2.3-kb open reading frame (ORF) preceded by potential ribosome-binding and promoter-like sequences. The ORF specified a putative protein of 775 amino acid residues with a calculated molecular weight of 87,533. The amino acid sequence deduced from the ORF exhibited significant similarity to that of the E. coli pfl gene.     

Listeriolysin O is essential for virulence of Listeria monocytogenes: direct evidence obtained by gene complementation.

The role of listeriolysin O in the intracellular multiplication of Listeria monocytogenes and, therefore, its pathogenicity was questioned through a genetic complementation study. A nonhemolytic mutant was generated by inserting a single copy of transposon Tn917 in the bacterial chromosome. This insertion was localized by DNA sequence analysis in hlyA, the gene coding for listeriolysin O. As was another mutant that we previously characterized, this mutant was avirulent in the mouse. It was transformed with a plasmid carrying only hlyA, able to replicate in L. monocytogenes, and stably maintained in vitro and in vivo. The complemented strain displayed a hemolytic phenotype identical to that of the wild-type strain and was fully virulent, therefore attributing a crucial role to listeriolysin O in virulence and excluding the hypothesis of a polar effect of the transposon insertion on genes adjacent to hlyA and possibly involved in virulence.     

Effects on virulence of mutations in a locus essential for hyaluronic acid capsule expression in group A streptococci.

Mucoid or highly encapsulated strains of group A streptococci have been associated both with unusually severe infections and with acute rheumatic fever. Previously, we described an acapsular mutant, TX4, derived from a mucoid M-type 18 strain of a group A streptococcus by transposon mutagenesis (M. R. Wessels, A. E. Moses, J. B. Goldberg, and T. J. DiCesare, Proc. Natl. Acad. Sci. USA 88:8317-8321, 1991). We now report studies further characterizing strain TX4 as well as an additional acapsular mutant, TX72. Strain TX4 was found to contain a 9.5-kb deletion of chromosomal DNA adjacent to the site of transposon Tn916 insertion. Cloned chromosomal DNA from TX4 flanking the transposon insertion site was used as a probe to demonstrate the presence of homologous regions in 11 of 11 wild-type group A streptococcal strains of various M protein types. A second acapsular mutant, TX72, had a single transposon insertion and had no apparent deletion of chromosomal DNA. The Tn916 insertion in TX72 was mapped to the hasA locus (encoding hyaluronate synthase), which lies within the chromosomal region deleted in TX4. Strain TX72 was avirulent in mice and sensitive to phagocytic killing in vitro. Transduction of either the insertion-deletion mutation from TX4 or the simple insertion mutation from TX72 to a type 24 group A streptococcus strain also resulted in loss of capsule expression, demonstrating that a homologous region of the chromosome controls capsule expression in another serotype of group A streptococci. We conclude that the hyaluronic acid capsule plays an important role in virulence and that a region of the chromosome essential for capsular polysaccharide expression is conserved among diverse group A streptococcal strains.