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Meningococcal internalization into human cells is likely to be a consequence of meningococcal adhesion to human epithelial and endothelial cells. Here, we identified three transposon mutants of Neisseria meningitidis that were primarily defective in the internalization of human brain microvascular endothelial cells (HBMEC), with insertions occurring in the gltT (a sodium-independent L-glutamate transporter) gene or its neighboring gene, NMB1964 (unknown function). NMB1964 was tentatively named gltM in this study because of the presence of a mammalian cell entry (MCE)-related domain in the deduced amino acid sequences. The null gltT-gltM N. meningitidis mutant was also defective in the internalization into human umbilical vein endothelial cells and the human lung carcinoma epithelial cell line A549, and the defect was suppressed by transcomplementation of the mutants with gltT+-gltM+genes. The intracellular survival of the gltT-gltM mutant in HBMEC was not largely different from that of the wild-type strain under our experimental conditions. Introduction of a1-bp deletion and amber or ochre mutations in gltT-gltM genes resulted in the loss of efficient internalization into HBMEC. The defect in meningococcal internalization into HBMEC and L-glutamate uptake in the gltT-gltM mutant were suppressed only in strains expressing both GltT and GltM proteins. The efficiency of meningococcal invasion to HBMEC decreased under L-glutamate-depleted conditions. Furthermore, ezrin, a key membrane-cytoskeleton linker, accumulated beneath colonies of the gltT+-gltM+N. meningitidis strain but not of the gltT-gltM mutant. These findings suggest that L-glutamate influx via the GltT-GltM L-glutamate ABC transporter serves as a cue for N. meningitidis internalization into host cells.

ABSTRACTThe lipooligosaccharide (LOS) of Neisseria meningitidiscan be decorated with phosphoethanolamine (PEA) at the 4′ position of lipid A and at the O-3 and O-6 positions of the inner core of the heptose II residue. The biological role of PEA modification in N. meningitidisremains unclear. During the course of our studies to elucidate the pathogenicity of the ST-2032 (invasive) meningococcal clonal group, disruption of lptA, the gene that encodes the PEA transferase for 4′ lipid A, led to a approximately 10-fold decrease in N. meningitidisadhesion to four kinds of human endothelial and epithelial cell lines at an multiplicity of infection of 5,000. Complementation of the lptAgene in a ΔlptAmutant restored wild-type adherence. By matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis, PEA was lost from the lipid A of the ΔlptAmutant compared to that of the wild-type strain. The effect of LptA on meningococcal adhesion was independent of other adhesins such as pili, Opc, Opa, and PilC but was inhibited by the presence of capsule. These results indicate that modification of LOS with PEA by LptA enhances meningococcal adhesion to human endothelial and epithelial cells in unencapsulated N. meningitidis.

ABSTRACTLyme disease Borreliaorganisms are highly invasive spirochetes that alternate between vertebrate and arthropod hosts and that establish chronic infections and elicit inflammatory reactions in mammals. Although progress has been made in the targeted mutagenesis of individual genes in infectious Borrelia burgdorferi, the roles of the vast majority of gene products in pathogenesis remain unresolved. In this study, we examined the feasibility of using transposon mutagenesis to identify infectivity-related factors in B. burgdorferi. The transformable, infectious strain 5A18 NP1 was transformed with the spirochete-adapted Himar1transposon delivery vector pMarGent to create a small library of 33 insertion mutants. Single mouse inoculations followed by culture of four tissue sites and serology were used to screen the mutants for infectivity phenotypes. Mutants that appeared attenuated (culture positive at some sites) or noninfectious (negative at all sites) and contained the virulence-associated plasmids lp25 and lp28-1 were examined in more extensive animal studies. Three of these mutants (including those with insertions in the putative fliG-1-encoded flagellar motor switch protein and the guaB-encoded IMP dehydrogenase) were noninfectious, whereas four clones appeared to exhibit reduced infectivity. Serological reactivity in VlsE enzyme-linked immunosorbent assays correlated with the assignment of mutants to the noninfectious or attenuated-infectivity groups. The results of this study indicate that random transposon mutagenesis of infectious B. burgdorferiis feasible and will be of value in studying the pathogenesis of Lyme disease Borrelia.

ABSTRACTPorphyromonas gingivalisis a crucial component of complex plaque biofilms that form in the oral cavity, resulting in the progression of periodontal disease. To elucidate the mechanism of periodontal biofilm formation, we analyzed the involvement of several genes related to the synthesis of polysaccharides in P. gingivalis. Gene knockout P. gingivalismutants were constructed by insertion of an ermF-ermAMcassette; among these mutants, the galEmutant showed some characteristic phenotypes involved in the loss of GalE activity. As expected, the galEmutant accumulated intracellular carbohydrates in the presence of 0.1% galactose and did not grow in the presence of galactose at a concentration greater than 1%, in contrast to the parental strain. Lipopolysaccharide (LPS) analysis indicated that the length of the O-antigen chain of the galEmutant was shorter than that of the wild type. It was also demonstrated that biofilms generated by the galEmutant had an intensity 4.5-fold greater than those of the wild type. Further, the galEmutant was found to be significantly susceptible to some antibiotics in comparison with the wild type. In addition, complementation of the galEmutation led to a partial recovery of the parental phenotypes. We concluded that the galEgene plays a pivotal role in the modification of LPS O antigen and biofilm formation in P. gingivalisand considered that our findings of a relationship between the function of the P. gingivalis galEgene and virulence phenotypes such as biofilm formation may provide clues for understanding the mechanism of pathogenicity in periodontal disease.

ABSTRACTThe OspE2 product of Shigellaspp., the expression of which is regulated by the mxiEgene, is secreted through a type III secretion system into host cells. We investigated the function of OspE2 of Shigella sonneiby using cultured epithelial cells. Cells invaded by an ospE2deletion mutant altered their morphology into the rounding shape, which was not due to cell death, whereas cells invaded by the wild-type strain kept their cell shape intact. The ospE2mutation did not affect initial cell entry and multiplication in cells, but the mutant formed smaller-than-normal plaques on cell monolayers, indicating a deficiency in cell-to-cell spread by the bacteria. An mxiEdeletion mutant also showed changes in cell morphology and deficiency in bacterial spread to adjacent cells. In cells invaded by the ospE2mutant, disturbance of actin stress fibers was prominent at 3 h after invasion. Analysis of OspE2 localization indicated that the OspE2 protein accumulated on focal contact-like structures in the infected host cells. These results suggest that colocalization of the OspE2 protein in the focal contacts of infected cells may function to maintain an intact cell morphology. The morphological change induced by invasion of the ospE2mutant may affect secondary bacterial transmission.

ABSTRACTWe constructed highly transformable and infectious Borrelia burgdorferiB31 by inactivating BBE02, a putative restriction-modification gene on the linear plasmid lp25. The low-passage-number B31 clones 5A4 (containing all plasmids) and 5A18 (lp28-4−lp56−) were used for this study, and BBE02 was disrupted by homologous recombination. The transformation efficiency with the shuttle vector pBSV2C03::gntΔkanwas increased from <1 to ∼10 colonies per μg of DNA for 5A4 and 5A4 BBE02::Kanrand from 14 to approximately 600 colonies per μg of DNA for 5A18 and 5A18 BBE02::Kanr. lp25, which is required for infectivity in mice, was retained in BBE02 mutants transformed with pBSV2C03::gntΔkan, but lp25 was not detected in transformants of the parental clones 5A4 and 5A18. BBE02 disruptants and pBSV2C03::gntΔkantransformants of these clones remained infectious in C3H/HeN mice, and the 50% infective doses of the BBE02 disruptants were <102organisms per mouse. The inactivation of BBE02 thus eliminates a transformation barrier for infectious B. burgdorferiB31 and will provide a valuable tool for studying the virulence factors of Lyme disease.

ABSTRACTOral streptococci play a large role in dental biofilm formation, and several types interact as early colonizers with the enamel salivary pellicle to form the primary biofilm, as well as to incorporate other bacteria on tooth surfaces. Interactions of surface molecules of individual streptococci with the salivary pellicle on the tooth surface have an influence on the etiological properties of an oral biofilm. To elucidate the molecular interactions of streptococci with salivary components, binding between surface protein (SspB and PAg) peptides of Streptococcus gordoniiand Streptococcus sobrinuswere investigated by utilizing BIAcore biosensor technology. The analogous peptide [change of T at position 400 to K in SspB(390-402), resulting in the SspB(390-T400K-402) peptide] from S. gordoniishowed the greatest response for binding to salivary components and inhibited the binding of Streptococcus sanguisby more than 50% in a competitive inhibition assay in a comparison with other SspB and PAg peptides. This peptide also bound to the high-molecular-weight protein complex of salivary components and the agglutinin (gp340/DMBT1) peptide (scavenger receptor cysteine-rich domain peptide 2 [SRCRP 2]). In addition, the SspB(390-T400K-402) peptide was visualized by two surface positive charges in connection with the positively charged residues, in which lysine was a key residue for binding. Therefore, the region containing lysine may have binding activity in S. gordoniiand S. sanguis, and the SRCRP 2 region may function as a receptor for the binding. These findings may provide useful information regarding the molecular mechanism of early biofilm formation by streptococci on tooth surfaces.

ABSTRACTYersinia pestisstrain Yreka was grown at 27 or 37°C, and the lipid A structures (lipid A-27°C and lipid A-37°C) of the respective lipopolysaccharides (LPS) were investigated by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. Lipid A-27°C consisted of a mixture of tri-acyl, tetra-acyl, penta-acyl, and hexa-acyl lipid A's, of which tetra-acyl lipid A was most abundant. Lipid A-37°C consisted predominantly of tri- and tetra-acylated molecules, with only small amounts of penta-acyl lipid A; no hexa-acyl lipid A was detected. Furthermore, the amount of 4-amino-arabinose was substantially higher in lipid A-27°C than in lipid A-37°C. By use of mouse and human macrophage cell lines, the biological activities of the LPS and lipid A preparations were measured via their abilities to induce production of tumor necrosis factor alpha (TNF-α). In both cell lines the LPS and the lipid A from bacteria grown at 27°C were stronger inducers of TNF-α than those from bacteria grown at 37°C. However, the difference in activity was more prominent in human macrophage cells. These results suggest that in order to reduce the activation of human macrophages, it may be more advantageous for Y. pestisto produce less-acylated lipid A at 37°C.

In Japan, more than 10% of streptococcal toxic shock-like syndrome (TSLS) cases have been caused by Streptococcus pyogenes M3/T3 isolates since the first reported TSLS case in 1992. Most M3/T3 isolates from TSLS or severe invasive infection cases during 1992 to 2001 and those from noninvasive cases during this period are indistinguishable in pulsed-field gel electropherograms. The longest fragments of these recent isolates were 300 kb in size, whereas those of isolates recovered during or before 1973 were 260 kb in size. These 260- and 300-kb fragments hybridized to each other, suggesting the acquisition of an about 40-kb fragment by the recent isolates. The whole part of the acquired fragment was cloned from the first Japanese TSLS isolate, NIH1, and its nucleotide sequence was determined. The 41,796-bp fragment is temperate phage phiNIH1.1, containing a new superantigen gene speL near its right attachment site. The C-terminal part of the deduced amino acid sequence of speL has 48 and 46% similarity with well-characterized erythrogenic toxin SpeC and the most potent superantigen, SmeZ-2, respectively. None of 10 T3 isolates recovered during or before 1973 has speL, whereas all of 18 M3/T3 isolates recovered during or after 1992 and, surprisingly, Streptococcus equi subsp. equi ATCC 9527 do have this gene. Though plaques could not be obtained from phiNIH1.1, its DNA became detectable from the phage particle fraction upon mitomycin C induction, showing that this phage is not defective. A horizontal transfer of the phage carrying speL may explain the observed change in M3/T3 S. pyogenes isolates in Japan.

ABSTRACTIn Japan, more than 10% of streptococcal toxic shock-like syndrome (TSLS) cases have been caused by Streptococcus pyogenesM3/T3 isolates since the first reported TSLS case in 1992. Most M3/T3 isolates from TSLS or severe invasive infection cases during 1992 to 2001 and those from noninvasive cases during this period are indistinguishable in pulsed-field gel electropherograms. The longest fragments of these recent isolates were 300 kb in size, whereas those of isolates recovered during or before 1973 were 260 kb in size. These 260- and 300-kb fragments hybridized to each other, suggesting the acquisition of an about 40-kb fragment by the recent isolates. The whole part of the acquired fragment was cloned from the first Japanese TSLS isolate, NIH1, and its nucleotide sequence was determined. The 41,796-bp fragment is temperate phage φNIH1.1, containing a new superantigen gene speLnear its right attachment site. The C-terminal part of the deduced amino acid sequence of speLhas 48 and 46% similarity with well-characterized erythrogenic toxin SpeC and the most potent superantigen, SmeZ-2, respectively. None of 10 T3 isolates recovered during or before 1973 has speL, whereas all of 18 M3/T3 isolates recovered during or after 1992 and, surprisingly, Streptococcus equisubsp. equiATCC 9527 do have this gene. Though plaques could not be obtained from φNIH1.1, its DNA became detectable from the phage particle fraction upon mitomycin C induction, showing that this phage is not defective. A horizontal transfer of the phage carrying speLmay explain the observed change in M3/T3 S. pyogenesisolates in Japan.