Metabolite Cross-Feeding Enhances Virulence in a Model Polymicrobial Infection

Microbes within polymicrobial infections often display synergistic interactions resulting in enhanced pathogenesis; however, the molecular mechanisms governing these interactions are not well understood. Development of model systems that allow detailed mechanistic studies of polymicrobial synergy is a critical step towards a comprehensive understanding of these infections in vivo. In this study, we used a model polymicrobial infection including the opportunistic pathogen Aggregatibacter actinomycetemcomitans and the commensal Streptococcus gordonii to examine the importance of metabolite cross-feeding for establishing co-culture infections. Our results reveal that co-culture with S. gordonii enhances the pathogenesis of A. actinomycetemcomitans in a murine abscess model of infection. Interestingly, the ability of A. actinomycetemcomitans to utilize L-lactate as an energy source is essential for these co-culture benefits. Surprisingly, inactivation of L-lactate catabolism had no impact on mono-culture growth in vitro and in vivo suggesting that A. actinomycetemcomitans L-lactate catabolism is only critical for establishing co-culture infections. These results demonstrate that metabolite cross-feeding is critical for A. actinomycetemcomitans to persist in a polymicrobial infection with S. gordonii supporting the idea that the metabolic properties of commensal bacteria alter the course of pathogenesis in polymicrobial communities

Porphyromonas gingivalis–dendritic cell interactions: consequences for coronary artery disease

An estimated 80 million US adults have one or more types of cardiovascular diseases. Atherosclerosis is the single most important contributor to cardiovascular diseases; however, only 50% of atherosclerosis patients have currently identified risk factors. Chronic periodontitis, a common inflammatory disease, is linked to an increased cardiovascular risk. Dendritic cells (DCs) are potent antigen presenting cells that infiltrate arterial walls and may destabilize atherosclerotic plaques in cardiovascular disease. While the source of these DCs in atherosclerotic plaques is presently unclear, we propose that dermal DCs from peripheral inflamed sites such as CP tissues are a potential source. This review will examine the role of the opportunistic oral pathogen Porphyromonas gingivalis in invading DCs and stimulating their mobilization and misdirection through the bloodstream. Based on our published observations, combined with some new data, as well as a focused review of the literature we will propose a model for how P. gingivalis may exploit DCs to gain access to systemic circulation and contribute to coronary artery disease. Our published evidence supports a significant role for P. gingivalis in subverting normal DC function, promoting a semimature, highly migratory, and immunosuppressive DC phenotype that contributes to the inflammatory development of atherosclerosis and, eventually, plaque rupture

Cardiovascular disease and the role of oral bacteria

In terms of the pathogenesis of cardiovascular disease (CVD) the focus has traditionally been on dyslipidemia. Over the decades our understanding of the pathogenesis of CVD has increased, and infections, including those caused by oral bacteria, are more likely involved in CVD progression than previously thought. While many studies have now shown an association between periodontal disease and CVD, the mechanisms underpinning this relationship remain unclear. This review gives a brief overview of the host-bacterial interactions in periodontal disease and virulence factors of oral bacteria before discussing the proposed mechanisms by which oral bacterial may facilitate the progression of CVD

The Number of Direct Repeats in hagA Is Variable among Porphyromonas gingivalis Strains

The coding sequence for the surface protein hemagglutinin A (HagA) of Porphyromonas gingivalis 381 has previously been shown to contain four direct 1.35-kb repeats, designated rep(HA). This study was performed to determine if the number of rep(HA) units in hagA is consistently 4 or if allelic polymorphism exists among strains and/or upon multiple passage of P. gingivalis. To this end, primers which were homologous to the regions directly 5′ and 3′ of the repeat domain in hagA were synthesized. PCR conditions which allowed amplification of the 8.4-kb repeat region between the primers in P. gingivalis 381 were established. Genomic DNA templates from 13 other P. gingivalis strains and 9 fresh clinical isolates from patients were analyzed under the same conditions as used above. Analysis of these PCR products demonstrated that the strains tested had different numbers (two to four) of rep(HA) units in the respective hagA genes. The PCR products of 8.4, 7.0, and 5.7 kb represent four, three, and two repeats, respectively. One strain from each group (381, four repeats; W83, three repeats; and AJW4, two repeats) was also tested to determine if the number of repeats remained invariant upon passaging onto solid medium. No variability in the number of repeats in hagA within a strain was detected after 18 passages. P. gingivalis 381 was chosen for further testing in a mouse abscess model to determine if conditions of in vivo growth would select for deletions or duplications of the repeated sequences. Five days after infection, no change in the number of repeats was detected in cells recovered from either nonimmunized or preimmunized mice. This data indicates an interstrain variability of the number of repeat units and hence a size variability of the HagA protein of P. gingivalis, but unlike some surface antigens of other pathogenic species, the number of repeats remains relatively stable given the conditions of growth tested here

Genomic comparison of invasive and rare non-invasive strains reveals Porphyromonas gingivalis genetic polymorphisms

Porphyromonas gingivalis strains are shown to invade human cells in vitro with different invasion efficiencies, varying by up to three orders of magnitude.We tested the hypothesis that invasion-associated interstrain genomic polymorphisms are present in P. gingivalis and that putative invasion-associated genes can contribute to P. gingivalis invasion.Using an invasive (W83) and the only available non-invasive P. gingivalis strain (AJW4) and whole genome microarrays followed by two separate software tools, we carried out comparative genomic hybridization (CGH) analysis.We identified 68 annotated and 51 hypothetical open reading frames (ORFs) that are polymorphic between these strains. Among these are surface proteins, lipoproteins, capsular polysaccharide biosynthesis enzymes, regulatory and immunoreactive proteins, integrases, and transposases often with abnormal GC content and clustered on the chromosome. Amplification of selected ORFs was used to validate the approach and the selection. Eleven clinical strains were investigated for the presence of selected ORFs. The putative invasion-associated ORFs were present in 10 of the isolates. The invasion ability of three isogenic mutants, carrying deletions in PG0185, PG0186, and PG0982 was tested. The PG0185 (ragA) and PG0186 (ragB) mutants had 5.1×103-fold and 3.6×103-fold decreased in vitro invasion ability, respectively.The annotation of divergent ORFs suggests deficiency in multiple genes as a basis for P. gingivalis non-invasive phenotype. Access the supplementary material to this article: Supplement, table (see Supplementary files under Reading Tools online)

Generating Isoform-Specific Antibodies: Lessons from the Nucleocytoplasmic Glycoprotein Skp1

Antibodies that discriminate protein isoforms differing by modifications at specific amino acids have revolutionized studies of their functions. Skp1 is a novel nucleocytoplasmic glycoprotein that is hydroxylated at proline-143 and then O-glycosylated by a pentasaccharide attached via a GlcNAcα1, 4(trans)- hydroxyproline linkage. Skp1 isoform-specific antibodies were successfully obtained by immunizing mice or rabbits with KLH-coupled synthetic peptides bearing either unmodified Pro, 4(trans)-hydroxyproline, or D-GlcNAcα1,4 (trans)-hydroxyproline, and screening with corresponding BSA-conjugates or by Western blotting toward a panel of Skp1 isoforms. Antibodies specific for Skp1 or HO-Skp1 were not found in exhaustive murine trials, yet monospecific polyclonal antibodies were readily achieved in rabbits without crossadsorption. In all cases, antibodies were specific at the protein but not the peptide level, which suggests that conformation comprises part of the basis for recognition and which should be considered when developing screening strategies