- Volume 144, Issue 11, 1998

SUMMARY: Entembacter aemgenes is among the five most frequently isolated nosocomial pathogens in France, and this bacterium also shows increasing multidrug resistance. In this study, various E. aerogenes strains isolated from hospital units were characterized for their outer-membrane proteins, antibiotic susceptibilities (inhibition diameters and MICs) and resistance mechanisms associated with modification of envelope permeability (porin alteration and active efflux). Diminished outer-membrane permeability due to porin alterations was found in conjunction with the expression of an enzymic barrier in resistant isolates. Interestingly, changes in the functional expression of porins appeared to play a special role in susceptibility to cefepime. An active efflux to quinolones was also identified. Simultaneous changes in envelope permeability, i.e. a porin deficiency (in) and an efflux mechanism (out), were clearly evident in two clinical strains.

SUMMARY: Adherence to host target cells is a critical step in establishing infection with the sexually transmitted pathogen Tiichomonas vaginalis. Four parasite surface proteins mediating attachment to vaginal epithelial cells have been identified. One surface protein, termed AP33, was characterized further to identify domains interactive with previously generated antibodies and with host surface sites. N- and C-terminal deletion subclones were generated and tested for reactivity with both mAb and rabbit antiserum against AP33, and were also examined for their ability to bind to host cells. Surprisingly, the rabbit antiserum known to inhibit cytoadherence recognized an epitope(s) contained within 72 residues in the N-terminal half of the protein. However, the mAb epitope was immunoreactive with a 28-amino-acid region near the C-terminus. Subsequent mapping of this region with overlapping peptides identified a nine-amino-acid sequence reactive with the mAb. Equally surprising, two domains interactive with host cell surfaces were identified at distinct parts of AP33: one in the N-terminal half of the protein, and the other within 24 residues in the C-terminal third. Further analysis of the C-terminal binding domain revealed that a peptide representing this area could inhibit T. vaginalis cytoadherence by 40%.

SUMMARY: There is evidence that the lipid A-associated proteins (LAPs) of enteric bacteria can induce the synthesis of interleukin 1 (IL-1) and therefore may be important virulence factors. Porphyromonas gingivdis is now recognized as a major pathogen in the chronic inflammatory periodontal diseases and it has previously been reported that a crude LAP fraction from this organism could induce IL-1 and interleukin 6 (IL-6) synthesis. In the present study the chemical and biological properties of the LAPs of this bacterium have been further characterized. Analysis by SDS-PAGE has shown that the LAPs comprise nine proteins of molecular masses 81,68,48,47,28,25,20,17 and 16 kDa. These LAPS, at concentrations as low as 100 ng mV, were shown to stimulate human gingival fibroblasts, human peripheral blood mononuclear cells and whole human blood t o produce the pro-inflammatory cytokine IL-6. The cytokine-inducing activity of the LAPs was reduced after heat-inactivation and trypsinization, suggesting that the activity was not due to contaminating LPS. To establish which proteins in this mixture were the active cytokine inducers, the LAPs were separated by electrophoresis on polyacrylamide gels. The majority of the activity was associated with the 17 kDa LAP. N-terminal sequence analysis demonstrated that this protein was homologous t o an internal region of a conserved adhesin domain contained within a family of P. gingivdis extracellular proteins including the RI protease, Lys-gingipain, porphypain and haemagglutinin A. In addition to a role in adherence, the adhesin domain(s) of these proteins may also have cytokine-inducing properties. Furthermore, it has also been shown that the previously observed degradation of cytokines by P. gingivelis may be attributable to the catalytic domain of the RI protease. Thus, different domains within the same molecule appear t o have opposing actions on pro-inflammatory cytokine levels and the balance between these two activities may influencethe cytokine status of the periodontiurn in patients with the common chronic inflammatory conditions known as the periodontal diseases.

SUMMARY: The population biology of Streptococcus pneumoniae is poorly understood. Most of the important issues could be addressed by the molecular characterization of large, well sampled populations from carriage and from the different manifestations of pneumococcal disease. The authors have therefore developed a pneumococcal multilocus sequence typing scheme and database by sequencing - 450 bp fragments of seven housekeeping loci from 295 isolates. The combination of alleles at the seven loci provided an allelic profile, or sequence type (ST), and the relatedness between isolates was obtained by constructing a dendrogram from the matrix of paiwvise differences between STs. The typing scheme was validated using pneumococci of known genetic relatedness and could resolve 6 billion STs. Among 274 isolates from recent cases of invasive pneumococcal disease in eight countries,143 STs were resolved, but 12 STs contained at least five isolates (range 5-21 isolates). The repeated recovery of indistinguishable isolates from invasive disease in different countries implies that these STs define strains with an increased capacity to cause invasive disease. The relationship between STs and serotypes suggested that, in the longer term, capsular genes have been distributed horizontally within the pneumococcal population, but in the short term, expansion of clones occurs with only occasional changes of serotype. The multilocus sequence typing scheme provides a powerful new approach to the characterization of pneumococci, since it provides molecular typing datathat are electronically portable between laboratories, and which can be used to probe aspects of the population and evolutionary biology of these organisms. A Web sitefor themolecular characterization of pneumococci by MLST is available (http://mlst.zoo.ox.ac.uk).

SUMMARY: Five independent fluorescent pseudomonad isolates originating from Antarctica were analysed for their pyoverdine systems. A pyoverdine-related siderotyping, which involved pyoverdine-induced growth stimulation, pyoverdine-mediated iron uptake, pyoverdine analysis by electrophoresis and isoelectric focusing, revealed three different pyoverdine-related siderotypes among the five isolates. One siderotype, including Pseudomonas fluorescens 1W and P. fluorescens lOcW, was identical to that of P. fluorescens ATCC 13525. Two other strains, P. fluorescens 9AW and Pseudomonas putida 9BW, showed identical pyoverdine-related behaviour t o each other, whereas the fifth strain, P. fluorescens 5lW, had unique features compared to the other strains or to a set of 12 fluorescent Pseudomonas strains used as comparison material. Elucidation of the structure of the pyoverdines produced by the Antarctic strains supported the accuracy of the siderotyping methodology by confirming that pyoverdines from strains 1W and 1OcW had the same structures as the P. fluorescens ATCC 13525 pyoverdine, whereas the 9AW and 9BW pyoverdines are probably identical with the pyoverdine of P. fluorescens strain 244. Pyoverdine from strain 51W appeared t o be a novel pyoverdine since its structure was different from all previously established pyoverdine structures. Together with the conclusion that the Antarctic Pseudomonas strains have no special features at the level of their pyoverdines and pyoverdine-mediated iron metabolism compared to worldwide strains, the present work demonstrates that siderotyping provides a rapid means of screening for novel pyoverdines.

SUMMARY: The siderophore pyochelin of Pseudomonas aeruginosa is derived from one molecule of salicylate and two molecules of cysteine. Two cotranscribed genes, pChEF8 encoding peptide synthetases have been identified and characterized. pchE was required for the conversion of salicylate to dihydroaeruginoate (Dha), the condensation product of salicylate and one cysteine residue and pchF was essential for the synthesis of pyochelin from Dha. The deduced PchE(156 kDa) and PchF (197 kDa) proteins had adenylation, thiolation and condensationkyclization motifs arranged as modules which are typical of those peptide synthetases forming thiazoline rings. The pchEF genes were coregulated with the pchDCBA operon, which provides enzymes for the synthesis (PchBA) and activation (PchD) of salicylate as well as a putative thioesterase (PchC). Expression of a translational pchf-'/acZ fusion was strictly dependent on the PchR regulator and was induced by extracellular pyochelin, the end product of the pathway. Iron replete conditions led t o Fur (ferric uptake regulator)-dependent repression of the pchE -laciZ fusion. A translational pchD-lacZ fusion was also positively regulated by PchR and pyochelin and repressed by Fur and iron. Thus, autoinduction by pyochelin (or ferric pyochelin) and repression by iron ensure a sensitive control of the pyochelin pathway in P. aeruginosa.

SUMMARY: The second replicator region of the native plasmid pTAVl of Paracoccus versutus has been identified thus proving the composite nature of this replicon. The minimal replicon designated pTAV320 (43 kb) was cloned and sequenced. pTAV320 encodes three putative proteins - RepA, RepB and RepC. This replicator region shows strong structural and functional similarity to mpABC-type rep1 icons found in several Agrobacterium and Rhizobium plasmids. The origin of replication appears to be localized within the coding sequence of the repC gene. RepC was shown t o be essential for replication. RepA and RepB were necessary for stable maintenance of the plasmid, which implies a role in active partitioning. The presence of the complete sequence of pTAV320 (in its non-replicative form) could stabilize in cis pTAV202, a rninireplicon derived from the other pTAVl replicator region. Deletions introduced into the mpC gene abolished the ‘stabilizing’ activity of pTAV320, suggesting that the centromere-like sequence, necessary for partitioning, might be localized within this gene. The two replicator regions of pTAV1 (pTAV320 and pTAV202) expressed incompatibility towards the parental plasmid but were compatible in trans in P. wersutus cells. The pTAV320 replicon can be maintained in several Paracoccus, Agrobacterium, Rhizobium and Rhodohcter strains in addition t o P. versutus.

SUMMARY: Ten Staphylococcus aureus mutants, defective in the starvation-induced stationary phase of growth were isolated from two independent Tn917-LTVI transposon insertion libraries and were designated suv as they had apparent-suryival defects. Seven of these mutants were defective under amino-acid-limiting conditions alone. Two mutants (suv-3 and suw-20) demonstrated lower plating efficiency when starved for glucose, phosphate or amino acids and one mutant (suv-11) had reduced plating efficiency after amino acid or glucose starvation. All of the mutants tested were as resistant to hydrogen peroxide assault as the parent, but six were more sensitive to low pH conditions. All the mutants were physically mapped on the 5. aureus chromosome using PFGE. Chromosomal DNA flanking the Tn917-LNI insertion sites was rescued by cloning into Escherichia coli. DNA sequence analysis resulted in theidentification of a number of transposon-disrupted ORFs encoding putative components such as superoxide dismutase (suv-I), haem A synthase (suv-3)# a component of the 505 response (suv-9) and hypoxanthine-guanine phosphoribosyltransferase (suv-20). The Tn917-LTVI insertion created lac2 transcriptional fusions for some of the stationary-phase loci. Expression analysis indicated that suv-4 was induced at mid-exponential phase, whereas suv-3 and suv-II were induced at the onset of stationary phase. The possible roles of these suv components in stationary-phase survival or recovery is discussed.

SUMMARY: Although the Gram-positive bacterium Streptococcus pyogenes (group A streptococcus) has been considered an extracellular pathogen which adheres to human mucosal epithelium, the streptococcus possesses invasive capacity for cultured human epithelial cells. This study provides genetic and functional evidence supporting the conclusion that protein F is capable of mediating entry of S. pyogenes into HeLa cells. Using 111916 insertion mutagenesis or an isogenic 5. pyogenes strain with a defined mutation in the gene encoding protein F (prtF), it was observed that the invasive capacity was affected by the levels of surface-exposed protein F, but not by those of M protein. In addition, heterologous expression of protein F on Enterococcus faecalis conferred upon the bacteria an efficient invasive phenotype. Several assays demonstrated that both the fibronectin-binding domains of protein F, UR and RD2, were involved in host-cell invasion. In addition, coinfection experiments of HeLa cells with 5. pyogenes and an Escherichia coli K-12 strain expressing an afimbrial adhesin AFA-I showed that the uptake of S. pyogenes did notpermit internalization of the E. coli cells.