The Murine Oral Metatranscriptome Reveals Microbial and Host Signatures of Periodontal Disease.

Periodontal disease is accompanied by alterations to cellular profiles and biological activities of both the subgingival microbiome and host tissues. Although significant progress has been made in describing the molecular basis of the homeostatic balance of host-commensal microbe interactions in health compared to the destructive imbalance in disease, particularly with respect to immune and inflammatory systems, few studies have attempted a comprehensive analysis in diverse host models. Here, we describe the development and application of a metatranscriptomic approach to analysis of host-microbe gene transcription in a murine periodontal disease model, based on oral gavage infection using Porphyromonas gingivalis in C57BL6/J mice. We generated 24 metatranscriptomic libraries from individual mouse oral swabs, representing health and disease. On average, 76% ± 11.7% reads in each sample belonged to the murine host genome and the remainder to the microbes. We found 3,468 (2.4% of the total) murine host transcripts differentially expressed between health and disease, of which 76% were overexpressed in periodontitis. Predictably, there were prominent alterations to genes and pathways linked with the host immune compartment in disease-the CD40 signaling pathway being the top enriched biological process in this data set. However, in addition, we observed significant alterations to other biological processes in disease, particularly cellular/metabolic processes and biological regulation. The number of differentially expressed microbial genes particularly indicated shifts in carbon metabolism pathways in disease with potential consequences for metabolic end-product formation. Together, these metatranscriptome data reveal marked changes between the gene expression patterns in both the murine host and microbiota, which may represent signatures of health and disease, providing the basis for future functional studies of prokaryotic and eukaryotic cellular responses in periodontal disease. In addition, the noninvasive protocol developed in this study will enable further longitudinal and interventionist studies of host-microbe gene expression networks.

Loss of Neutrophil Homing to the Periodontal Tissues Modulates the Composition and Disease Potential of the Oral Microbiota.

Periodontal disease is considered to arise from an imbalance in the interplay between the host and its commensal microbiota, characterized by inflammation, destructive periodontal bone loss, and a dysbiotic oral microbial community. The neutrophil is a key component of defense of the periodontium: defects in their number or efficacy of function predisposes individuals to development of periodontal disease. Paradoxically, neutrophil activity, as part of a deregulated inflammatory response, is considered an important element in the destructive disease process. In this investigation, we examined the role the neutrophil plays in the regulation of the oral microbiota by analysis of the microbiome composition in mice lacking the CXCR2 neutrophil receptor required for recruitment to the periodontal tissues. A breeding protocol was employed that ensured that only the oral microbiota of wild-type (CXCR2+/+) mice was transferred to subsequent generations of wild-type, heterozygote, and homozygote littermates. In the absence of neutrophils, the microbiome undergoes a significant shift in total load and composition compared to when normal levels of neutrophil recruitment into the gingival tissues occur, and this is accompanied by a significant increase in periodontal bone pathology. However, transfer of the oral microbiome of CXCR2-/- mice into germfree CXCR2+/+ mice led to restoration of the microbiome to the wild-type CXCR2+/+ composition and the absence of pathology. These data demonstrate that the composition of the oral microbiome is inherently flexible and is governed to a significant extent by the genetics and resultant phenotype of the host organism.

The P. gingivalis Autocitrullinome Is Not a Target for ACPA in Early Rheumatoid Arthritis.

Rheumatoid arthritis (RA), a chronic inflammatory disease affecting primarily the joints, is frequently characterized by the presence of autoimmune anticitrullinated protein antibodies (ACPA) during preclinical stages of disease and accumulation of hypercitrullinated proteins in arthritic joints. A strong association has been reported between RA and periodontal disease, and Porphyromonas gingivalis, a known driver of periodontitis, has been proposed as the microbial link underlying this association. We recently demonstrated P. gingivalis-mediated gut barrier breakdown and exacerbation of joint inflammation during inflammatory arthritis. In the present study, we investigated another potential role for P. gingivalis in RA etiopathogenesis, based on the generation of ACPA through the activity of a unique P. gingivalis peptidylarginine deiminase (PPAD) produced by this bacterium, which is capable of protein citrullination. Using a novel P. gingivalis W50 PPAD mutant strain, incapable of protein citrullination, and serum from disease-modifying antirheumatic drug-naïve early arthritis patients, we assessed whether autocitrullinated proteins in the P. gingivalis proteome serve as cross-activation targets in the initiation of ACPA production. We found no evidence for patient antibody activity specific to autocitrullinated P. gingivalis proteins. Moreover, deletion of PPAD did not prevent P. gingivalis-mediated intestinal barrier breakdown and exacerbation of disease during inflammatory arthritis in a murine model. Together, these findings suggest that the enzymatic activity of PPAD is not a major virulence mechanism during early stages of inflammatory arthritis.

Horizontal and Vertical Transfer of Oral Microbial Dysbiosis and Periodontal Disease.

One of the hallmark features of destructive periodontal disease, well documented over the last 50 y, is a change to the quantitative and qualitative composition of the associated microbiology. These alterations are now generally viewed as transformational shifts of the microbial populations associated with health leading to the emergence of bacterial species, which are only present in low abundance in health and a proportionate decrease in the abundance of others. The role of this dysbiosis of the health associated microbiota in the development of disease remains controversial: is this altered microbiology the driving agent of disease or merely a consequence of the altered environmental conditions that invariably accompany destructive disease? In this work, we aimed to address this controversy through controlled transmission experiments in the mouse in which a dysbiotic oral microbiome was transferred either horizontally or vertically into healthy recipient mice. The results of these murine studies demonstrate conclusively that natural transfer of the dysbiotic oral microbiome from a periodontally diseased individual into a healthy individual will lead to establishment of the dysbiotic community in the recipient and concomitant transmission of the disease phenotype. The inherent resilience of the dysbiotic microbial community structure in diseased animals was further demonstrated by analysis of the effects of antibiotic therapy on periodontally diseased mice. Although antibiotic treatment led to a reversal of dysbiosis of the oral microbiome, in terms of both microbial load and community structure, dysbiosis of the microbiome was reestablished following cessation of therapy. Collectively, these data suggest that an oral dysbiotic microbial community structure is stable to transfer and can act in a similar manner to a conventional transmissible infectious disease agent with concomitant effects on pathology. These findings have implications to our understanding of the role of microbial dysbiosis in the development and progression of human periodontal disease.

Hemin binding by Porphyromonas gingivalis strains is dependent on the presence of A-LPS.

Porphyromonas gingivalis is a Gram-negative black pigmenting anaerobe that is unable to synthesize heme [Fe(II)-protoporphyrin IX] or hemin [Fe(III)-protoporphyrin IX-Cl], which are important growth/virulence factors, and must therefore derive them from the host. Porphyromonas gingivalis expresses several proteinaceous hemin-binding sites, which are important in the binding/transport of heme/hemin from the host. It also synthesizes several virulence factors, namely cysteine-proteases Arg- and Lys-gingipains and two lipopolysaccharides (LPS), O-LPS and A-LPS. The gingipains are required for the production of the black pigment, µ-oxo-bisheme {[Fe(III)PPIX]2 O}, which is derived from hemoglobin and deposited on the bacterial cell-surface leading to the characteristic black colonies when grown on blood agar. In this study we investigated the role of LPS in the deposition of µ-oxo-bisheme on the cell-surface. A P. gingivalis mutant defective in the biosynthesis of Arg-gingipains, namely rgpA/rgpB, produces brown colonies on blood agar and mutants defective in Lys-gingipain (kgp) and LPS biosynthesis namely porR, waaL, wzy, and pg0129 (α-1, 3-mannosyltransferase) produce non-pigmented colonies. However, only those mutants lacking A-LPS showed reduced hemin-binding when cells in suspension were incubated with hemin. Using native, de-O-phosphorylated and de-lipidated LPS from P. gingivalis W50 and porR strains, we demonstrated that hemin-binding to O-polysaccharide (PS) and to the lipid A moiety of LPS was reduced compared with hemin-binding to A-PS. We conclude that A-LPS in the outer-membrane of P. gingivalis serves as a scaffold/anchor for the retention of µ-oxo-bisheme on the cell surface and pigmentation is dependent on the presence of A-LPS.

Genetic analysis of mechanisms of multidrug resistance in a clinical isolate of Bacteroides fragilis.

This study investigated the mechanisms of multidrug resistance (MDR) in an isolate of Bacteroides fragilis (WI1) from a patient with anaerobic sepsis. The MDR of WI1 affected susceptibility to beta-lactams, clindamycin, fluoroquinolones, metronidazole and tetracycline. In addition to its 5.31-Mb chromosome, WI1 possessed two low-copy-number plasmids, pHagl (5.6 kb) and pHag2 (9.9 kb), that were absent from B. fragilis NCTC 9343. Restriction digestion with EcoRV, HindIII and SstI, combined with DNA sequencing, revealed that pHAG2 contained a tet(Q) gene at base position 3689 that resided on the conjugative transposon CTn341. Genes cfiA (encoding a metallo-beta-lactamase) and erm(F) (encoding a macrolide-lincosamide-streptogramin B resistance determinant) were also found in WI1, but were absent from B. fragilis NCTC 9343. Nitrocefin hydrolysis revealed that WI1 had high beta-lactamase activity. Sequencing of the gyrA quinolone resistance-determining region revealed a mutation causing a Ser82 --> Phe substitution, and comparative quantitative real-time RT-PCR revealed that the cfiA, erm(F) and tet(Q) genes were all expressed in WI1. In addition, the resistance-nodulation-division efflux pump genes bmeB9 and bmeB15 were significantly over-expressed (12.30 +/- 0.42-fold and 3541.1 +/- 95.4-fold, respectively), and the efflux pump inhibitors carbonyl cyanide m-chlorophenylhydrazone and reserpine significantly increased the susceptibility of the isolate to several unrelated antibiotics (p <0.005). These data suggested that WI1 was highly multidrug-resistant because of the additive effects of chromosome- and plasmid-encoded resistance determinants.

Critical pathways in microbial virulence.

The virulence of a microbe represents a combination of complex factors including the agent's transmissibility and the severity of the disease associated with infection and is also significantly influenced by the susceptibility of the colonized host. Virulence factors may be defined as those products of the organism which are required to complete the various stages of the life cycle leading to pathology in the host. In this review, we examine some of the approaches which have been adopted in other fields of infectious disease in order to categorically identify virulence factors using a classical genetics approach with relevant models or human subjects. The absence of an accurate experimental model for periodontal disease means that our understanding of the microbial virulence determinants and pathways in this disease remains hypothetical and based largely on observations in vitro. However, factors which enable the organism to persist in spite of the elevated immune and inflammatory pressure at sites of disease are liable to be critical. Periodontal bacterial genomics is liable to make a significant impact on the field through an increased appreciation of the role of gene acquisition and gene loss in the evolution of periodontal bacteria and of the consequences of strain variation in gene content on virulence potential.

Oro-dental bacteria in various atherosclerotic arteries.

Chlamydia pneumoniae DNA has been detected in at least 40% of all major arteries affected by atherosclerosis, but several other microorganisms have also been detected. In this study, diseased vessels were evaluated for the presence of the DNA of seven oro-dental bacteria and two nonoral bacteria. A polymerase chain reaction technique was employed using primer pairs based on 16S rRNA genes. Of 32 specimens tested, 10 (31.2%) were DNA positive: seven for Actinobacillus actinomycetemcomitans and three for Prevotella intermedia. The DNA was found in specimens from the aorta and the iliac, internal mammary and coronary arteries. Eleven (35.4%) of 31 specimens had been shown previously to be positive for Chlamydia pneumoniae DNA. A mixture of chlamydiae and oro-dental bacteria was found in three cases. These findings may have implications for antibiotic prophylaxis of coronary heart disease if directed solely at Chlamydia pneumoniae.

Attenuation of the virulence of Porphyromonas gingivalis by using a specific synthetic Kgp protease inhibitor.

The Arg- and Lys-gingipains of Porphyromonas gingivalis are important virulence determinants in periodontal disease and may correspond to targets for immune- or drug-based treatment strategies. In this investigation we aimed to determine which of these enzymes represents the most promising molecular target for protease inhibitor-based therapy and to examine the effectiveness of the resultant compound in a murine virulence assay. Isogenic mutants with mutations in rgpA and rgpB (encoding Arg-gingipains) and in kgp (encoding Lys-gingipain) and a double mutant with mutations in rgpA and rgpB were prepared by using P. gingivalis W50. The virulence of these mutants indicated that Kgp is a promising drug target. Combinatorial chemistry was used to define the optimal substrate of Kgp, and from this information a specific slowly reversible inhibitor with a nanomolar K(i) was designed and synthesized. Growth of P. gingivalis W50 in the presence of this compound resembled the phenotype of the kgp isogenic mutant; in both instances bacterial colonies failed to form pigment on blood agar, and only poor growth was obtained in a defined medium containing albumin as the sole protein source. Furthermore, pretreatment of the wild-type organism with the Kgp inhibitor led to a significant reduction in virulence in the murine assay. These data emphasize the conclusion that Kgp is an important factor for both nutrition and virulence of P. gingivalis and that inhibitors of this enzyme may have therapeutic potential for the control of P. gingivalis infections. Protease inhibitors may be a potentially novel class of antimicrobial agents with relevance to the control of other bacterial pathogens.

Molecular mediators of Porphyromonas gingivalis-induced T-cell apoptosis.

Porphyromonas gingivalis produces virulence factors which can modify the molecular and cellular components of the host immune response. In the present work we investigated the role of specific virulence factors from P. gingivalis in the induction of apoptosis in Jurkat T cells. P. gingivalis culture supernatants mimicked the effect of butyric acid on T-cell apoptosis and this effect was associated with an increase in histone H4 acetylation. A role for proteases was excluded in experiments which demonstrated that neither protease inhibitors nor use of P. gingivalis mutants defective in protease synthesis had any effect on the stimulation of T-cell apoptosis in this system.

Cysteine proteases of Porphyromonas gingivalis.

The cysteine proteases of Porphyromonas gingivalis are extracellular products of an important etiological agent in periodontal diseases. Many of the in vitro actions of these enzymes are consistent with the observed deregulated inflammatory and immune features of the disease. They are significant targets of the immune responses of affected individuals and are viewed by some as potential molecular targets for therapeutic approaches to these diseases. Furthermore, they appear to represent a complex group of genes and protein products whose transcriptional and translational control and maturation pathways may have a broader relevance to virulence determinants of other persistent bacterial pathogens of human mucosal surfaces. As a result, the genetics, chemistry, and virulence-related properties of the cysteine proteases of P. gingivalis have been the focus of much research effort over the last ten years. In this review, we describe some of the progress in their molecular characterization and how their putative biological roles, in relation to the in vivo growth and survival strategies of P. gingivalis, may also contribute to the periodontal disease process.

Identification of ragAB as a temperature-regulated operon of Porphyromonas gingivalis W50 using differential display of randomly primed RNA.

Porphyromonas gingivalis is a gram-negative, black-pigmented anaerobe that has been associated with advanced periodontal disease. The genome of P. gingivalis has the potential to produce a number of virulence determinants including proteases, hemagglutinins, hemolysin, invasion-associated proteins, and products of the pathogenicity island ragAB; however, little is known about how their expression is controlled. Periodontal pockets experience a higher temperature during inflammation, and this elevated temperature may influence the pathogenicity of P. gingivalis by changing its patterns of gene expression. In this study, RNA has been isolated from cells of P. gingivalis grown to steady state at temperatures of 37, 39, and 41 degrees C under hemin excess conditions (pH 7.0) in a chemostat. The RNA was subjected to PCR amplification following reverse transcription, using various combinations of randomly selected oligonucleotide primers. Reproducible RNA fingerprints have been obtained; however, differences were demonstrated in the RNA profiles of cells grown at the three temperatures, indicating differences in gene expression. Several PCR fragments were isolated that appeared to represent temperature-regulated genes. The nucleotide sequence of one of these has been identified as part of the ragAB locus, which codes for both a 55-kDa immunodominant antigen (RagB) and a homologue of the family of TonB-linked outer membrane receptors (RagA). These data indicate that expression of ragAB may be modulated in response to changes in temperature and that this may suggest a mechanism of evading the host response in the inflamed periodontal pocket.

Variable carbohydrate modifications to the catalytic chains of the RgpA and RgpB proteases of Porphyromonas gingivalis W50.

Proteases of Porphyromonas gingivalis are considered to be important virulence determinants of this periodontal bacterium. Several biochemical isoforms of arginine-specific proteases are derived from rgpA and rgpB. HRgpA is a heterodimer composed of the catalytic alpha chain noncovalently associated with a beta adhesin chain derived from the C terminus of the initial full-length translation product. The catalytic alpha chain is also present as a monomer (RgpA) either free in solution or associated with membranes. rgpB lacks the coding region for the adhesin domain present in rgpA and yields only monomeric forms (RgpB) which again may be soluble or membrane associated. In this study, the catalytic chains of this unusual group of enzymes are shown to be differentially modified by the posttranslational addition of carbohydrate. A monoclonal antibody (MAb 1B5) raised to the monomeric RgpA did not react with the corresponding recombinant RgpA alpha chain expressed in Escherichia coli but was immunoreactive with P. gingivalis lipopolysaccharide. MAb 1B5 also reacted with the membrane-associated forms of RgpA and RgpB but not with the heterodimeric HRgpA and the soluble form of RgpB. RgpA treated with denaturants was capable of binding to MAb 1B5 whereas treatment with periodate abolished this binding, suggesting the presence of carbohydrate residues within the epitope. Chemical deglycosylation abolished immunoreactivity with MAb 1B5 and caused a approximately 30% reduction in the size of the membrane-associated enzymes. Monosaccharide analysis of HRgpA and RgpA demonstrated 2.1 and 14.4%, respectively, carbohydrate by weight of protein. Furthermore, distinct differences were detected in their monosaccharide compositions, indicating that these protease isoforms are modified not only to different extents but also with different sugars. The variable nature of these additions may have a significant effect on the structure, stability, and immune recognition of these protease glycoproteins.

Effect of temperature on growth, hemagglutination, and protease activity of Porphyromonas gingivalis.

Bacteria persisting in periodontal pockets are exposed to elevated temperatures during periods of inflammation. Temperature is an environmental factor that can modulate gene expression. Consequently, in the present study we examined the effect of temperature on the expression of virulence determinants by the periodontopathogen, Porphyromonas gingivalis. P. gingivalis W50 was grown in a complex medium under hemin excess at pH 7.0 and at a constant temperature of either 37, 39, or 41 degrees C; cultures were monitored for protease and hemagglutinin activity. P. gingivalis grew well at all three temperatures. An increase in growth temperature from 37 to 39 degrees C resulted in a 65% reduction in both total arginine- and lysine-specific activities (P < 0.01). A further rise in growth temperature to 41 degrees C led to even greater reductions in arginine-specific (82%; P < 0.001) and lysine-specific (73%; P < 0. 01) activities. These reductions were also associated with an altered distribution of individual arginine-specific enzyme isoforms. At 41 degrees C, there was a disproportionate reduction in the level of the heterodimeric RI protease, which also contains adhesin domains. The reduction also correlated with a markedly diminished hemagglutination activity of cells, especially in those grown at 41 degrees C, and a reduced immunoreactivity with a monoclonal antibody which recognizes gene products involved in hemagglutination. Thus, as the environmental temperature increased, P. gingivalis adopted a less aggressive phenotype, while retaining cell population levels. The coordinate down-regulation of virulence gene expression in response to an environmental cue linked to the intensity of the host inflammatory response is consistent with the clinically observed cyclical nature of disease progression in periodontal diseases.

A 55-kilodalton immunodominant antigen of Porphyromonas gingivalis W50 has arisen via horizontal gene transfer.

A 55-kDa outer membrane protein of Porphyromonas gingivalis W50 is a significant target of the serum immunoglobulin G antibody response of periodontal disease patients and hence may play an important role in host-bacterium interactions in periodontal disease. The gene encoding the 55-kDa antigen (ragB, for receptor antigen B) was isolated on a 9.5-kb partial Sau3AI fragment of P. gingivalis W50 chromosomal DNA in pUC18 by immunoscreening with a monoclonal antibody to this antigen. The 1.6-kb open reading frame (ORF) encoding RagB was located via subcloning and nested-deletion analysis. Sequence analysis demonstrated the presence of an upstream 3.1-kb ORF (ragA) which is cotranscribed with ragB. A number of genetic characteristics suggest that the ragAB locus was acquired by a horizontal gene transfer event. These include a significantly reduced G+C content relative to that of the P. gingivalis chromosome (42 versus 48%) and the presence of mobility elements flanking this locus in P. gingivalis W50. Furthermore, Southern blotting and PCR analyses showed a restricted distribution of this locus in laboratory and clinical isolates of this bacterium. The association of ragAB+ P. gingivalis with clinical status was examined by PCR analysis of subgingival samples. ragAB+ was not detected in P. gingivalis-positive shallow pockets from periodontal disease patients but was present in 36% of the P. gingivalis-positive samples from deep pockets. These data suggest that the ragAB locus was acquired by certain P. gingivalis strains via horizontal gene transfer and that the acquisition of this locus may facilitate the survival of these strains at sites of periodontal destruction.

The rag locus of Porphyromonas gingivalis: a novel pathogenicity island.

Previous studies in our laboratories of the serum IgG antibody response of periodontal patients have demonstrated the presence of an immunodominant surface antigen (Mr 55 kDa) in the outer membrane of Porphyromonas gingivalis W50. Genetic analysis of this antigen revealed that the corresponding gene forms part of a small operon which may have arisen via horizontal gene transfer into the genome of this strain. On the basis of sequence homology, the 55 kDa antigen (RagB) and the product of a cotranscribed gene (RagA) may act in concert at the surface of the bacterium to facilitate active transport, mediated through the periplasmic spanning protein, TonB, or form part of a signal transduction system in this organism. The rag locus is present in only a proportion of P. gingivalis laboratory strains and clinical isolates. Analysis of the distribution of ragB in subgingival samples by PCR demonstrated that rag+ P. gingivalis are more frequently detected in deep periodontal pockets than shallow sites in periodontal patients. These findings indicate that the rag genes may influence the virulence potential of P. gingivalis strains which harbour this locus and may thus be considered a novel pathogenicity island. Furthermore, horizontal gene transfer between organisms in subgingival plaque may represent a significant force in the evolution of these bacteria with ramifications for both diagnosis and targeted treatment of periodontal disease.

Molecular genetics and nomenclature of proteases of Porphyromonas gingivalis.

The strategies used by bacterial pathogens to establish and maintain themselves in the host represent one of the fundamental aspects of microbial pathogenesis. Characterization of these strategies and the underlying molecular machinery offers new opportunities both to our understanding of how organisms cause disease in susceptible individuals and to the development of novel therapeutic measures designed to undermine or interfere with these determinants of successful survival. With respect to the microbial aetiology of the periodontal diseases, a growing body of evidence suggests that the proteolytic enzymes of Porphyromonas gingivalis represent key survival and, by extrapolation, virulence determinants of this periodontal bacterium. This in turn has led to international efforts to characterize these enzymes at the gene and protein level. Approximately 20 protease genes of P. gingivalis with different names and accession numbers have been deposited in the gene databases and a correspondingly heterogeneous nomenclature system is employed for the products of these genes in the literature. However, it is evident, through comparison of these gene sequences and through gene inactivation studies, that the genetic structure of the proteases of this organism, particularly those with specificity for arginyl and lysyl peptide bonds, is less complicated than originally thought. The major extracellular and surface associated arginine specific protease activity is encoded by 2 genes which we recommend be designated rgpA and rgpB (arg-gingipains A & B). Similarly we recommend that the gene encoding the major lysine specific protease activity is designated kgp (lys-gingipain). These three genes, which account for all the extracellular/surface arginine and lysine protease activity in P. gingivalis, belong to a family of sequence-related proteases and haemagglutinins.

Maturation of the arginine-specific proteases of Porphyromonas gingivalis W50 is dependent on a functional prR2 protease gene.

The prpR1 of Porphyromonas gingivalis codes for three distinct enzymes with specificity for arginyl peptide bonds termed RI, RIA, and RIB. These three isoforms comprise the majority of the extracellular, arginine-specific protease activity in P. gingivalis W50. RI is a heterodimer in which the catalytic alpha chain is noncovalently associated with a second chain involved in adherence phenomena. RIA and RIB are both monomeric species. RIA represents the free alpha chain, and RIB is a highly posttranslationally modified form of the alpha chain which is exclusively vesicle or membrane associated and migrates as a diffuse band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In previous studies, insertional inactivation of the prpR1 demonstrated that arginine-specific protease activity can also arise from a closely related second gene, prR2. In the present work, the prR2 was insertionally inactivated in P. gingivalis W50 in order to establish the contribution of this locus to the arginine-specific protease activity of this periodontal bacterium. Loss of prR2 function had several effects on prpR1-derived enzymes. First, the total Arg-X activity was reduced by approximately 50% relative to that of the parent strain. The reduction in total activity was a consequence of decreased concentrations of the monomeric enzymes derived from the prpR1, while the heterodimeric enzyme, RI, was unaffected by this mutation. Second, the chromatographic behavior of both the soluble and vesicle- or membrane-associated monomeric enzymes was radically different from the behavior of RIA and RIB from the parent strain. Finally, the vesicle- or membrane-associated enzyme in the prR2 mutant strain lacked the extensive posttranslational additions which are found on RIB in P. gingivalis W50. These data suggest that the product(s) of the prR2 plays a significant role in the maturation pathway of prpR1-derived enzymes, and this may contribute to the coconservation of these two genes in P. gingivalis.

The Tla protein of Porphyromonas gingivalis W50: a homolog of the RI protease precursor (PrpRI) is an outer membrane receptor required for growth on low levels of hemin.

The prpR1 gene of Porphyromonas gingivalis W50 encodes the polyprotein precursor (PrpRI) of an extracellular arginine-specific protease. PrpRI is organized into four distinct domains (pro, alpha, beta, and gamma) and is processed to a heterodimeric protease (RI) which comprises the alpha and beta components in a noncovalent association. The alpha component contains the protease active site, whereas the beta component appears to have a role in adherence and hemagglutination processes. DNA sequences homologous to the coding region for the RI beta component are present at multiple loci on the P. gingivalis chromosome and may represent a family of related genes. In this report, we describe the cloning, sequence analysis, and characterization of one of these homologous loci isolated in plasmid pJM7. The 6,041-bp P. gingivalis DNA fragment in pJM7 contains a major open reading frame of 3,291 bp with coding potential for a protein with an Mr 118,700. An internal region of the deduced sequence (V304 to N768) shows 98% identity to the beta domain of PrpRI, and the recombinant product of pJM7 is immunoreactive with an antibody specific to the RI beta component. The N terminus of the deduced sequence has regional similarity to TonB-linked receptors which are frequently involved in periplasmic translocation of hemin, iron, colicins, or vitamin B12 in other bacteria. We have therefore designated this gene tla (TonB-linked adhesin). In contrast to the parent strain, an isogenic mutant of P. gingivalis W50 in which the tla was insertionally inactivated was unable to grow in medium containing low concentrations of hemin (<2.5 mg liter(-1)), and hemin-depleted cells of this mutant failed to respond to hemin in an agar diffusion plate assay. These data suggest a role for this gene product in hemin acquisition and utilization. Furthermore, the mutant produced significantly less arginine- and lysine-specific protease activities than the parent strain, indicating that there may be a regulatory relationship between tla and other members of this gene family.

The prpR1 and prR2 arginine-specific protease genes of Porphyromonas gingivalis W50 produce five biochemically distinct enzymes.

The arginine-specific protease activity of Porphyromonas gingivalis is considered to be an important factor in the pathogenic potential of this organism in destructive periodontal disease. Multiple forms of closely related Arg-x proteases are present in the culture supernatants of P. gingivalis W50. RI is a heterodimer (alpha/beta) in which the catalytic alpha chain is associated with a second beta chain which functions as a haemagglutinin. RIA is a single-chain enzyme (alpha) and RIB is a highly post-translationally lipid-modified enzyme (LPS-alpha) with reduced solubility compared to the other two forms. The N-terminal sequence of the alpha chain of all three forms is identical, suggesting that all these enzymes may arise by differential processing of the prpR1 (protease polyprotein for RI). In the present study we constructed a prpR1- strain of P. gingivalis W50 by insertional gene inactivation and characterized the residual extracellular Arg-x protease activity of the resulting mutant. Loss of prpR1 expression led to the abolition of RI, RIA and RIB but the total Arg-x activity in the supernatant of this strain was reduced by only c. 66%. The remaining activity was composed of two novel forms of Arg-x protease (RIIA and RIIB) which appeared to be structurally and kinetically almost identical to RIA and RIB, respectively, except for two amino acid differences in the N-terminus at position 8 (Q-->E) and position 17 (A-->P) and with respect to their stability to high pH. Confirmation that RIIA and RIIB are the products of a homologous locus (prR2) was obtained by cloning and sequencing the prR2 which showed the predicted substitutions in the deduced translation. These data indicate that RI, RIA and RIB are produced by prpR1 expression and a maturation pathway which can give rise to a dimer and an unmodified- or LPS-modified catalytic monomer. Furthermore, RIIA and RIIB, the products of prR2, are exported into the culture supernatant in the absence of prpR1 expression and these forms may also contribute to the pathogenic potential of this organism in destructive disease.