Yixin Shi

Genetic analyses of proteolysis, hemoglobin binding, and hemagglutination of Porphyromonas gingivalis: Construction of mutants with a combination of rgpA, rgpB, kgp, and hagA

Porphyromonas gingivalis produces arginine-specific cysteine proteinase (Arg-gingipain, RGP) and lysine-specific cysteine proteinase (Lys-gingipain, KGP) in the extracellular and cell-associated forms. Two separate genes (rgpA and rgpB) and a single gene (kgp) have been found to encode RGP and KGP, respectively. We constructed rgpA rgpB kgp triple mutants by homologous recombination with cloned rgp and kgp DNA interrupted by drug resistance gene markers. The triple mutants showed no RGP or KGP activity in either cell extracts or culture supernatants. The culture supernatants of the triple mutants grown in a rich medium had no proteolytic activity toward bovine serum albumin or gelatin derived from human type I collagen. Moreover, the mutants did not grow in a defined medium containing bovine serum albumin as the sole carbon/energy source. These results indicate that the proteolytic activity of P. gingivalis toward bovine serum albumin and gelatin derived from human type I collagen appears to be attributable to RGP and KGP. The hemagglutinin gene hagA of P. gingivalis possesses the adhesin domain regions responsible for hemagglutination and hemoglobin binding that are also located in the C-terminal regions of rgpA and kgp. ArgpA kgp hagA triple mutant constructed in this study exhibited no hemagglutination using sheep erythrocytes or hemoglobin binding activity, as determined by a solid-phase binding assay with horseradish peroxidase-conjugated human hemoglobin, indicating that the adhesin domains seem to be particularly important for P. gingivalis cells to agglutinate erythrocytes and bind hemoglobin, leading to heme acquisition.

PhoP-regulated Salmonella resistance to the antimicrobial peptides magainin 2 and polymyxin B

In Salmonella enterica, the PhoP–PhoQ two‐component system governs resistance to structurally different antimicrobial peptides including the alpha‐helical magainin 2, the β‐sheet defensins and the cyclic lipopeptide polymyxin B. To identify the PhoP‐regulated determinants mediating peptide resistance, we prepared a plasmid library from a phoP mutant, introduced it into a phoP mutant and selected for magainin‐resistant clones. One of the clones harboured the PhoP‐activated ugtL gene, deletion of which rendered Salmonella susceptible to magainin 2 and polymyxin B, but not defensin HNP‐1. We established that ugtL encodes an inner membrane protein that promotes the formation of monophosphorylated lipid A in the lipopolysaccharide. Inactivation of both ugtL and the regulatory gene pmrA, which controls lipid A modifications required for resistance to polymxyin B (but not to magainin 2) and is post‐transcriptionally activated by the PhoP–PhoQ system, resulted in a strain that was as susceptible to polymyxin B as a phoP mutant. The most frequently recovered clone harboured the yqjA gene, which we show is PhoP regulated and required for resistance to magainin 2 but not to polymyxin B or defensin HNP‐1. Our results indicate that different PhoP‐mediated modifications in lipid A are necessary for resistance to different antimicrobial peptides.

Construction and characterization of a nonpigmented mutant of Porphyromonas gingivalis: cell surface polysaccharide as an anchorage for gingipains

A nonpigmented mutant of Porphyromonas gingivalis was constructed by using transposon mutagenesis. The mutant possessed the transposon DNA at the novel gene porR. Gene targeted mutagenesis revealed that porR was responsible for pigmentation. The porR gene shared similarities with genes of the degT family, the products of which are now considered to be transaminases involved in biosynthesis of sugar portions of cell-surface polysaccharides and aminoglycosides. The porR mutant showed a pleiotropic phenotype: delayed maturation of fimbrillin, preferential presence of Rgp and Kgp proteinases in culture supernatants, and no haemagglutination. The porR mutant had altered phenol extractable polysaccharide compared to the porR(+) sibling strain. A mAb, 1B5, that reacts with sugar portions of P. gingivalis cell surface polysaccharide and membrane-type Rgp proteinase showed no reaction with the cell lysates of the porR mutant. These results indicate that porR is involved in biosynthesis of cell surface polysaccharide that may function as an anchorage for Rgp, Kgp, haemagglutinins and the haemoglobin receptor protein.

A novel peroxiredoxin of the plant Sedum lineare is a homologue of Escherichia coli bacterioferritin co-migratory protein (Bcp).

We cloned a gene encoding a 17-kDa protein from a cDNA library of the plant Sedum lineare and found that its deduced amino acid sequence showed similarities to those of Escherichia coli bacterioferritin co-migratory protein (Bcp) and its homologues, which comprise a discrete group associated with the peroxiredoxin (Prx) family. Studies of the recombinant 17-kDa protein produced in E. coli cells revealed that it actually had a thioredoxin-dependent peroxidase activity, the hallmark of the Prx family. PrxQ, as we now designate the 17-kDa protein, had two cysteine residues (Cys-44 and Cys-49) well conserved among proteins of the Bcp group. These two cysteines were demonstrated to be essential for the thioredoxin-dependent peroxidase activity by analysis of mutant proteins, suggesting that these residues are involved in the formation of an intramolecular disulphide bond as an intermediate in the reaction cycle. Expression of PrxQ suppressed the hypersensitivity of an E. coli bcp mutant to peroxides, indicating that it might exert an antioxidant activity in vivo.

Porphyromonas gingivalis-induced platelet aggregation in plasma depends on Hgp44 adhesin but not Rgp proteinase

Evidence from recent epidemiological studies suggests a link between periodontal infections and increased risk of atherosclerosis and related cardiovascular and cerebrovascular events in human subjects. One of the major pathogens of periodontitis, Porphyromonas gingivalis, has the ability to aggregate human platelets in platelet‐rich plasma (PRP). Mechanism of P. gingivalis‐induced platelet aggregation in PRP was investigated. Proteinase inhibitors toward Arg‐gingipain (Rgp) and Lys‐gingipain (Kgp) did not suppress P. gingivalis‐induced platelet aggregation in PRP, whereas the Rgp inhibitor markedly inhibited P. gingivalis‐induced platelet aggregation using  washed  platelets.  Mutant  analysis  revealed  that P. gingivalis‐induced platelet aggregation in PRP depended on Rgp‐, Kgp‐ and haemagglutinin A (HagA)‐encoding genes that intragenically coded for adhesins such as Hgp44. Hgp44 adhesin on the bacterial cell surface, which was processed by Rgp and Kgp proteinases, was essential for P. gingivalis‐induced platelet aggregation in PRP. P. gingivalis cell‐reactive IgG in plasma, and FcγRIIa receptor and to a lesser extent GPIbα receptor on platelets were found to be a prerequisite for P. gingivalis‐induced platelet aggregation in PRP. These results reveal a novel mechanism of platelet aggregation by P. gingivalis.

The CpxR/CpxA Two-component System Up-regulates Two Tat-dependent Peptidoglycan Amidases to Confer Bacterial Resistance to Antimicrobial Peptide

We demonstrate that the twin arginine translocation (Tat) system contributes to bacterial resistance to cationic antimicrobial peptides (CAMPs). Our results show that a deletion at the tatC gene, which encodes a subunit of the Tat complex, caused Salmonella and Escherichia coli to become susceptible to protamine. We screened chromosomal loci that encode known and predicted Tat-dependent proteins and found that two N-acetylmuramoyl-l-alanine amidases, encoded by amiA and amiC, elevated bacterial resistance to protamine and α-helical peptides magainin 2 and melittin but not to β-sheet defensin HNP-1 and lipopeptide polymyxin B. Genetic analysis suggests that transcription of both amiA and amiC loci in Salmonella is up-regulated by the CpxR/CpxA two-component system when nlpE is overexpressed. A footprinting analysis reveals that CpxR protein can interact with amiA and amiC promoters at the CpxR box, which is localized between the predicted −10 and −35 regions but present on different strands in these two genes. In addition, our results show that activation of the CpxR/CpxA system can facilitate protamine resistance because nlpE overexpression elevates this resistance in the wild-type strain but not the cpxR deletion mutant. Thus, we uncover a new transcriptional regulation pathway in which the Cpx envelope stress response system modulates the integrity of the cell envelope in part by controlling peptidoglycan amidase activity, which confers bacterial resistance to protamine and α-helical CAMPs. Our studies have important implications for understanding transcriptional regulation of peptidoglycan metabolism and also provide new insights into the role of the bacterial envelope in CAMP resistance.

Human Lactoferrin Binds and Removes the Hemoglobin Receptor Protein of the Periodontopathogen Porphyromonas gingivalis

Porphyromonas gingivalis possesses a hemoglobin receptor (HbR) protein on the cell surface as one of the major components of the hemoglobin utilization system in this periodontopathogenic bacterium. HbR is intragenically encoded by the genes of an arginine-specific cysteine proteinase (rgpA), lysine-specific cysteine proteinase (kgp), and a hemagglutinin (hagA). Here, we have demonstrated that human lactoferrin as well as hemoglobin have the abilities to bind purified HbR and the cell surface of P. gingivalis through HbR. The interaction of lactoferrin with HbR led to the release of HbR from the cell surface of P. gingivalis. This lactoferrin-mediated HbR release was inhibited by the cysteine proteinase inhibitors effective to the cysteine proteinases of P. gingivalis. P. gingivalis could not utilize lactoferrin for its growth as an iron source and, in contrast, lactoferrin inhibited the growth of the bacterium in a rich medium containing hemoglobin as the sole iron source. Lactoferricin B, a 25-amino acid-long peptide located at the N-lobe of bovine lactoferrin, caused the same effects onP. gingivalis cells as human lactoferrin, indicating that the effects of lactoferrin might be attributable to the lactoferricin region. These results suggest that lactoferrin has a bacteriostatic action on P. gingivalis by binding HbR, removing it from the cell surface, and consequently disrupting the iron uptake system from hemoglobin.

Ferritin from the obligate anaerobe Porphyromonas gingivalis: purification, gene cloning and mutant studies

Porphyromonas gingivalis is an obligate anaerobe that utilizes haem, transferrin and haemoglobin efficiently as sources of iron for growth, and has the ability to store haem on its cell surface, resulting in black pigmentation of colonies on blood agar plates. However, little is known about intracellular iron storage in this organism. Ferritin is one of the intracellular iron-storage proteins and may also contribute to the protection of organisms against oxidative stresses generated by intracellular free iron. A ferritin-like protein was purified from P. gingivalis and the encoding gene (ftn) was cloned from chromosomal DNA using information on its amino-terminal amino acid sequence. Comparison of the amino acid sequence deduced from the nucleotide sequence of ftn with those of known ferritins and bacterioferritins identified the protein as a ferritin and positioned it between proteins from the Proteobacteria and Thermotogales. The P. gingivalis ferritin was found to contain non-haem iron, thus confirming its identity. Construction and characterization of a P. gingivalis ferritin-deficient mutant revealed that the ferritin was particularly important for the bacterium to survive under iron-depleted conditions (both haemin and transferrin starvation), indicating that intracellular iron is stored in ferritin regardless of the iron source and that the iron stored in ferritin is utilized under iron-restricted conditions. However, the ferritin appeared not to contribute to protection against oxidative stresses caused by peroxides and atmospheric oxygen.

The CpxR/CpxA two-component regulatory system up-regulates the multidrug resistance cascade to facilitate Escherichia coli resistance to a model antimicrobial peptide.

Background: The CpxR/CpxA system facilitates E. coli resistance to antimicrobial peptides. However, many CpxR-dependent mechanisms remain elusive. Results: The CpxR/CpxA system activates mar transcription, subsequently facilitating E. coli resistance to protamine via tripartite multidrug transporters. Conclusion: A CpxR/CpxA-dependent regulatory cascade controls MarRA for CAMP resistance in response to aromatic metabolites. Significance: This is novel regulatory mechanism controlling bacterial resistance to CAMPs and antibiotics. A genome-wide susceptibility assay was used to identify specific CpxR-dependent genes that facilitate Escherichia coli resistance to a model cationic antimicrobial peptide, protamine. A total of 115 strains from the Keio Collection, each of which contained a deletion at a demonstrated or predicted CpxR/CpxA-dependent locus, were tested for protamine susceptibility. One strain that exhibited high susceptibility carried a deletion of tolC, a gene that encodes the outer membrane component of multiple tripartite multidrug transporters. Concomitantly, two of these efflux systems, AcrAB/TolC and EmrAB/TolC, play major roles in protamine resistance. Activation of the CpxR/CpxA system stimulates mar transcription, suggesting a new regulatory circuit that enhances the multidrug resistance cascade. Tripartite multidrug efflux systems contribute to bacterial resistance to protamine differently from the Tat system. DNase I footprinting analysis demonstrated that the CpxR protein binds to a sequence located in the −35 and −10 regions of mar promoter. This sequence resembles the consensus CpxR binding site, however, on the opposite strand. aroK, a CpxR-dependent gene that encodes a shikimate kinase in the tryptophan biosynthesis pathway, was also found to facilitate protamine resistance. Specific aromatic metabolites from this pathway, such as indole, can stimulate expression of well studied CpxR-dependent genes degP and cpxP, which are not components of the tripartite multidrug transporters. Thus, we propose a novel mechanism for E. coli to modulate resistance to protamine and likely other cationic antimicrobial peptides in which the CpxR/CpxA system up-regulates mar transcription in response to specific aromatic metabolites, subsequently stimulating the multidrug resistance cascade.

A dual-signal regulatory circuit activates transcription of a set of divergent operons in Salmonella typhimurium

We present a molecular mechanism for signal transduction that activates transcription of the SlyA regulon in Salmonella typhimurium. We demonstrate that SlyA mediates transcriptional activation in response to guanosine tetraphosphate, ppGpp, according to the following observations: (i) in vivo transcription of SlyA-dependent genes is repressed when ppGpp is absent; this transcription can be restored by overproducing SlyA; (ii) in vivo dimerization and binding of SlyA to the target promoter are facilitated in the presence of ppGpp; and (iii) in vitro SlyA binding to the target promoter is enhanced when ppGpp is supplemented. Thus, ppGpp must be the cytoplasmic component that stimulates SlyA regulatory function by interacting directly with this regulator in Salmonella. This signaling domain, integrated by the PhoP/PhoQ 2-component system that activates slyA transcription by sensing Mg2+, forms feedforward loops that regulate chromosomal loci identified through a motif search over the S. typhimurium genome. Many such loci are divergent operons, each formed by 2 neighboring genes in which transcription of these 2 loci proceeds in opposite directions. Both genes, however, are controlled by PhoP and SlyA through a single shared PhoP box and SlyA box present in their intergenic regions. A substitution in either box sequence causes a simultaneous cessation of transcription of a divergent operon, pagD-pagC, equivalent to the phenotype in a phoP or slyA mutant. We also identified several chromosomal loci that possess pagC-type genes without the cognate pagD-type genes. Therefore, our results provide a molecular basis for the understanding of SlyA-dependent phenotypes associated with Salmonella virulence.