A. Wilson Aruni

Filifactor alocis Has Virulence Attributes That Can Enhance Its Persistence under Oxidative Stress Conditions and Mediate Invasion of Epithelial Cells by Porphyromonas gingivalis

ABSTRACT Filifactor alocis, a Gram-positive anaerobic rod, is one of the most abundant bacteria identified in the periodontal pockets of periodontitis patients. There is a gap in our understanding of its pathogenicity and ability to interact with other periodontal pathogens. To evaluate the virulence potential of F. alocis and its ability to interact with Porphyromonas gingivalis W83, several clinical isolates of F. alocis were characterized. F. alocis showed nongingipain protease and sialidase activities. In silico analysis revealed the molecular relatedness of several virulence factors from F. alocis and P. gingivalis. In contrast to P. gingivalis, F. alocis was relatively resistant to oxidative stress and its growth was stimulated under those conditions. Biofilm formation was significantly increased in coculture. There was an increase in adherence and invasion of epithelial cells in coculture compared with P. gingivalis or F. alocis monocultures. In those epithelial cells, endocytic vesicle-mediated internalization was observed only during coculture. The F. alocis clinical isolate had an increased invasive capacity in coculture with P. gingivalis compared to the ATCC 35896 strain. In addition, there was variation in the proteomes of the clinical isolates compared to the ATCC 35896 strain. Hypothetical proteins and those known to be important virulence factors in other bacteria were identified. These results indicate that F. alocis has virulence properties that may enhance its ability to survive and persist in the periodontal pocket and may play an important role in infection-induced periodontal disease.

Proteome variation among Filifactor alocis strains

Filifactor alocis, a Gram‐positive anaerobic rod, is now considered one of the marker organisms associated with periodontal disease. Although there was heterogeneity in its virulence potential, this bacterium was shown to have virulence properties that may enhance its ability to survive and persist in the periodontal pocket. To gain further insight into a possible mechanism(s) of pathogenesis, the proteome of F. alocis strains was evaluated. Proteins including several proteases, neutrophil‐activating protein A and calcium‐binding acid repeat protein, were identified in F. alocis. During the invasion of HeLa cells, there was increased expression of several of the genes encoding these proteins in the potentially more virulent F. alocis D‐62D compared to F. alocis ATCC 35896, the type strain. A comparative protein in silico analysis of the proteome revealed more cell wall anchoring proteins in the F. alocis D‐62D compared to F. alocis ATCC 35896. Their expression was enhanced by coinfection with Porphyromonas gingivalis. Taken together, the variation in the pathogenic potential of the F. alocis strains may be related to the differential expression of several putative virulence factors.

Proteome Analysis of Coinfection of Epithelial Cells with Filifactor alocis and Porphyromonas gingivalis Shows Modulation of Pathogen and Host Regulatory Pathways

ABSTRACT Changes in periodontal status are associated with shifts in the composition of the bacterial community in the periodontal pocket. The relative abundances of several newly recognized microbial species, including Filifactor alocis, as-yet-unculturable organisms, and other fastidious organisms have raised questions on their impact on disease development. We have previously reported that the virulence attributes of F. alocis are enhanced in coculture with Porphyromonas gingivalis. We have evaluated the proteome of host cells and F. alocis during a polymicrobial infection. Coinfection of epithelial cells with F. alocis and P. gingivalis strains showed approximately 20% to 30% more proteins than a monoinfection. Unlike F. alocis ATCC 35896, the D-62D strain expressed more proteins during coculture with P. gingivalis W83 than with P. gingivalis 33277. Proteins designated microbial surface component-recognizing adhesion matrix molecules (MSCRAMMs) and cell wall anchor proteins were highly upregulated during the polymicrobial infection. Ultrastructural analysis of the epithelial cells showed formation of membrane microdomains only during coinfection. The proteome profile of epithelial cells showed proteins related to cytoskeletal organization and gene expression and epigenetic modification to be in high abundance. Modulation of proteins involved in apoptotic and cell signaling pathways was noted during coinfection. The enhanced virulence potential of F. alocis may be related to the differential expression levels of several putative virulence factors and their effects on specific host cell pathways.

The Biofilm Community: Rebels with a Cause

Oral biofilms are some of the most complex and diverse ecosystems developed by successive colonization of more than 600 bacterial taxa. Development starts with the attachment of early colonizers such as Actinomyces species and oral streptococci on the acquired pellicle and tooth enamel. These bacteria not only adhere to the tooth’s surface, but also interact with each other and lay foundation for attachment of bridging colonizers such as Fusobacterium nucleatum followed by late colonizers including the red complex species Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola, the founders of periodontal disease. As the biofilm progresses from supragingival sites to subgingival sites, the environment changes from aerobic to anaerobic, thus favoring the growth of mainly Gram-negative obligate anaerobes while restricting the growth of the early Gram-positive facultative aerobes. Microbes present at the supragingival level are mainly related to gingivitis and root caries, whereas subgingival species advance the destruction of teeth supporting tissues, and thus cause periodontitis. This review summarizes our present understanding and recent developments on the characteristic features of supra- and subgingival biofilms, interaction between different genera and species of bacteria constituting these biofilms, and draws our attention to the role of some of the recently discovered members of the oral community.

Nitrate reductase activity of bacteria in saliva of term and preterm infants.

The salivary glands of adults concentrate nitrate from plasma into saliva where it is converted to nitrite by bacterial nitrate reductases. Nitrite can play a beneficial role in adult gastrointestinal and cardiovascular physiology. When nitrite is swallowed, some of it is converted to nitric oxide (NO) in the stomach and may then exert protective effects in the gastrointestinal tract and throughout the body. It has yet to be determined either when newborn infants acquire oral nitrate reducing bacteria or what the effects of antimicrobial therapy or premature birth may be on the bacterial processing of nitrate to nitrite. We measured nitrate and nitrite levels in the saliva of adults and both preterm and term human infants in the early weeks of life. We also measured oral bacterial reductase activity in the saliva of both infants and adults, and characterized the species of nitrate reducing bacteria present. Oral bacterial conversion of nitrate to nitrite in infants was either undetectable or markedly lower than the conversion rates of adults. No measurable reductase activity was found in infants within the first two weeks of life, despite the presence of oral nitrate reducing bacteria such as Actinomyces odontolyticus, Veillonella atypica, and Rothia mucilaginosa. We conclude that relatively little nitrite reaches the infant gastrointestinal tract due to the lack of oral bacterial nitrate reductase activity. Given the importance of the nitrate-nitrite-NO axis in adults, the lack of oral nitrate-reducing bacteria in infants may be relevant to the vulnerability of newborns to hypoxic stress and gastrointestinal tract pathologies.

VimA-Dependent Modulation of Acetyl Coenzyme A Levels and Lipid A Biosynthesis Can Alter Virulence in Porphyromonas gingivalis

ABSTRACT The Porphyromonas gingivalis VimA protein has multifunctional properties that can modulate several of its major virulence factors. To further characterize VimA, P. gingivalis FLL406 carrying an additional vimA gene and a vimA-defective mutant in a different P. gingivalis genetic background were evaluated. The vimA-defective mutant (FLL451) in the P. gingivalis ATCC 33277 genetic background showed a phenotype similar to that of the vimA-defective mutant (FLL92) in the P. gingivalis W83 genetic background. In contrast to the wild type, gingipain activity was increased in P. gingivalis FLL406, a vimA chimeric strain. P. gingivalis FLL451 had a five times higher biofilm-forming capacity than the parent strain. HeLa cells incubated with P. gingivalis FLL92 showed a decrease in invasion, in contrast to P. gingivalis FLL451 and FLL406, which showed increases of 30 and 40%, respectively. VimA mediated coenzyme A (CoA) transfer to isoleucine and reduced branched-chain amino acid metabolism. The lipid A content and associated proteins were altered in the vimA-defective mutants. The VimA chimera interacted with several proteins which were found to have an LXXTG motif, similar to the sorting motif of Gram-positive organisms. All the proteins had an N-terminal signal sequence with a putative sorting signal of L(P/T/S)X(T/N/D)G and two unique signatures of EXGXTX and HISXXGXG, in addition to a polar tail. Taken together, these observations further confirm the multifunctional role of VimA in modulating virulence possibly through its involvement in acetyl-CoA transfer and lipid A synthesis and possibly by protein sorting.

regT can modulate gingipain activity and response to oxidative stress in Porphyromonas gingivalis

Recombinant VimA protein can interact with the gingipains and several other proteins that may play a role in its biogenesis in Porphyromonas gingivalis. In silico analysis of PG2096, a hypothetical protein that was shown to interact with VimA, suggests that it may have environmental stress resistance properties. To further evaluate the role(s) of PG2096, the predicted open reading frame was PCR amplified from P. gingivalis W83 and insertionally inactivated using the ermF-ermAM antibiotic-resistance cassette. One randomly chosen PG2096-defective mutant created by allelic exchange and designated FLL205 was further characterized. Under normal growth conditions at 37 °C, Arg-X and Lys-X gingipain activities in FLL205 were reduced by approximately 35 % and 21 %, respectively, compared to the wild-type strain. However, during prolonged growth at an elevated temperature of 42 °C, Arg-X activity was increased by more than 40 % in FLL205 in comparison to the wild-type strain. In addition, the PG2096-defective mutant was more resistant to oxidative stress when treated with 0.25 mM hydrogen peroxide. Taken together these results suggest that the PG2096 gene, designated regT (regulator of gingipain activity at elevated temperatures), may be involved in regulating gingipain activity at elevated temperatures and be important in oxidative stress resistance in P. gingivalis.