Sequential action of R- and K-specific gingipains of Porphyromonas gingivalis in the generation of the heam-containing pigment from oxyhaemoglobin

Smalley, John W.; Birss, Andrew J.; Szmigielski, Borys; Potempa, Jan

doi:10.1016/j.abb.2007.05.011

Cited by 46 publications

(87 citation statements)

References 21 publications

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“…S3A in the supplemental material, oxyhemoglobin enhanced the growth of bacilli relative to the level of growth for the BSMonly control, albeit at lower levels than methemoglobin. Interestingly, hemoglobin bound to carbon monoxide (CO-hemoglobin) also enhanced growth, despite the fact that the CO-hemoglobin (as well as the oxyhemoglobin) versions of hemoglobin are known to be more resistant to degradation (36,37). All hemoglobins in this assay were confirmed to be in the expected oxidation state by absorbance spectroscopy (see Fig.…”

Section: Resultsmentioning

confidence: 62%

Bacillus anthracis Overcomes an Amino Acid Auxotrophy by Cleaving Host Serum Proteins

et al. 2015

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Bacteria sustain an infection by acquiring nutrients from the host to support replication. The host sequesters these nutrients as a growth-restricting strategy, a concept termed "nutritional immunity." Historically, the study of nutritional immunity has centered on iron uptake because many bacteria target hemoglobin, an abundant circulating protein, as an iron source. Left unresolved are the mechanisms that bacteria use to attain other nutrients from host sources, including amino acids. We employed a novel medium designed to mimic the chemical composition of human serum, and we show here that Bacillus anthracis, the causative agent of anthrax disease, proteolyzes human hemoglobin to liberate essential amino acids which enhance its growth. This property can be traced to the actions of InhA1, a secreted metalloprotease, and extends to at least three other serum proteins, including serum albumin. The results suggest that we must also consider proteolysis of key host proteins to be a way for bacterial pathogens to attain essential nutrients, and we provide an experimental framework to determine the host and bacterial factors involved in this process. IMPORTANCEThe mechanisms by which bacterial pathogens acquire nutrients during infection are poorly understood. Here we used a novel defined medium that approximates the chemical composition of human blood serum, blood serum mimic (BSM), to better model the nutritional environment that pathogens encounter during bacteremia. Removing essential amino acids from BSM revealed that two of the most abundant proteins in blood-hemoglobin and serum albumin-can satiate the amino acid requirement for Bacillus anthracis, the causative agent of anthrax. We further demonstrate that hemoglobin is proteolyzed by the secreted protease InhA1. These studies highlight that common blood proteins can be a nutrient source for bacteria. They also challenge the historical view that hemoglobin is solely an iron source for bacterial pathogens. There are three broad, necessary, and common elements inherent in most if not all bacterial infections of vertebrate hosts. They are (i) the initial entry of the bacterial invader and adherence to host tissues, (ii) the production of virulence factors or toxins (usually proteins) that subvert host innate and acquired immune defense mechanisms, and (iii) the acquisition and construction of biomolecules to drive cellular metabolism for the production of progeny. There has been intense investigation, and knowledge of the biochemistry, cell biology, and contribution to disease of a diverse array of bacterial adhesins and virulence factors (points i and ii identified above) is growing. However, our knowledge of nutrient acquisition during pathogenesis has lagged, perhaps because it is viewed as being a necessary and somewhat mundane process that may lack the elegant sophistication observed with virulence factors. Considering that all systemic infections are absolutely dependent on the replication of bacterial cells, identifying the mechanisms by which ba...

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Section: Resultsmentioning

confidence: 62%

Bacillus anthracis Overcomes an Amino Acid Auxotrophy by Cleaving Host Serum Proteins

et al. 2015

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“…Rgp is known to process the precursor form of Kgp to yield the mature, catalytically active form of the enzyme (17). In addition, Rgp converts oxyhemoglobin to methemoglobin, which is more readily degraded by Kgp to release free heme (30). Therefore, we propose that the PG_0264 mutant is nonpigmented at least in part due to its decreased expression of rgpA and therefore a smaller population of catalytically active Kgp proteins and lower levels of the methemoglobin substrate for Kgp.…”

Section: Discussionmentioning

confidence: 99%

Using Tn-seq To Identify Pigmentation-Related Genes of Porphyromonas gingivalis: Characterization of the Role of a Putative Glycosyltransferase

et al. 2017

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Cellular pigmentation is an important virulence factor of the oral pathogen Porphyromonas gingivalis. Pigmentation has been associated with many bacterial functions, including but not limited to colonization, maintaining a local anaerobic environment by binding oxygen molecules, and defense against reactive oxygen species (ROS) produced by immune cells. Pigmentation-associated loci identified to date have involved lipopolysaccharide, fimbriae, and heme acquisition and processing. We utilized a transposon mutant library of P. gingivalis strain ATCC 33277 and screened for pigmentation-defective colonies using massively parallel sequencing of the transposon junctions (Tn-seq) to identify genes involved in pigmentation. Transposon insertions at 235 separate sites, located in 67 genes and 15 intergenic regions, resulted in altered pigmentation: 7 of the genes had previously been shown to be involved in pigmentation, while 75 genes and intergenic regions had not. To further confirm identification, we generated a smaller transposon mutant library in P. gingivalis strain W83 and identified pigment mutations in several of the same loci as those identified in the screen in ATCC 33277 but also eight that were not identified in the ATCC 33277 screen. PGN_0361/ PG_0264, a putative glycosyltransferase gene located between two tRNA synthetase genes and adjacent to a miniature inverted-repeat transposable element, was identified in the Tn-seq screen and then verified through targeted deletion and complementation. Deletion mutations in PGN_0361/PG_0264 glycosyltransferase abolish pigmentation, modulate gingipain protease activity, and alter lipopolysaccharide. The mechanisms of involvement in pigmentation for other loci identified in this study remain to be determined, but our screen provides the most complete survey of genes involved in pigmentation to date. IMPORTANCE P. gingivalis has been implicated in the onset and progression of periodontal disease. One important virulence factor is the bacterium's ability to produce pigment. Using a transposon library, we were able to identify both known and novel genes involved in pigmentation of P. gingivalis. We identified a glycosyltransferase, previously not associated with pigmentation, that is required for pigmentation and determined its mechanism of involvement. A better understanding of the genes involved in pigmentation may lead to new insights into the complex mechanisms involved in this important virulence characteristic and could facilitate development of novel therapeutics.

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“…galE, rplD, and gapA were used as control genes. was not observed, the suppressed formation of -oxo bisheme may be explained by the fact that either RgpA or RgpB, or both, with Kgp activity is required by P. gingivalis to produce -oxo bisheme (45). The surface-accumulated hemin is transported into a bacterial cell to be utilized.…”

Section: Vol 55 2011 Antibacterial Action Of Poly(p) On P Gingivalmentioning

confidence: 99%

Antibacterial Action of Polyphosphate on Porphyromonas gingivalis

Moon

Park

Lee

2011

Antimicrob Agents Chemother

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Polyphosphate [poly(P)] has antibacterial activity against various Gram-positive bacteria. In contrast, Gram-negative bacteria are generally resistant to poly(P). Here, we describe the antibacterial characterization of poly(P) against a Gram-negative periodontopathogen, Porphyromonas gingivalis. The MICs of pyrophosphate (Na 4 P 2 O 7 ) and all poly(P) (Na n ؉ 2 P n O 3n ؉ 1 ; n ‫؍‬ 3 to 75) tested for the bacterium by the agar dilution method were 0.24% and 0.06%, respectively. Orthophosphate (Na 2 HPO 4 ) failed to inhibit bacterial growth. Poly-P75 was chosen for further study. In liquid medium, 0.03% poly-P75 was bactericidal against P. gingivalis irrespective of the growth phase and inoculum size, ranging from 10 5 to10 9 cells/ml. UV-visible spectra of the pigments from P. gingivalis grown on blood agar with or without poly-P75 showed that poly-P75 reduced the formation of -oxo bisheme by the bacterium. Poly-P75 increased hemin accumulation on the P. gingivalis surface and decreased energy-driven uptake of hemin by the bacterium. The expression of the genes encoding hemagglutinins, gingipains, hemin uptake loci, chromosome replication, and energy production was downregulated, while that of the genes related to iron storage and oxidative stress was upregulated by poly-P75. The transmission electron microscope showed morphologically atypical cells with electron-dense granules and condensed nucleoid in the cytoplasm. Collectively, poly(P) is bactericidal against P. gingivalis, in which hemin/heme utilization is disturbed and oxidative stress is increased by poly(P).Inorganic polyphosphate [poly(P)] is a ubiquitous compound found in bacteria, fungi, algae, plants, and animals. The poly(P) found in the organisms is a chain of a few or many hundreds of phosphate (P i ) residues linked by high-energy phosphoanhydride. It performs varied functions in bacteria: it can serve as an ATP source and substitute, it is a strong chelator of metal ions and thus can regulate the levels of the ions in the cells, it is a channel for DNA entry, and it is a regulator that contributes to bacterial resistance and survival under stress and stringent conditions (18). Therefore, intracellular poly(P) is considered a virulence factor of microorganisms.In contrast, exogenous poly(P) has attracted considerable attention as an antimicrobial agent, since it can prevent spoilage of food (29, 32), and it is listed as a GRAS (generally recognized as safe) food additive by the FDA. Poly(P) inhibits the growth of various Gram-positive bacteria, such as Staphylococcus aureus (14,17,22,35,52), Listeria monocytogenes (37, 52), Sarcina lutea (35), Bacillus cereus, and Lactobacillus, and of fungi, such as Aspergillus flavus (17,28). Concerning oral bacteria, mutans streptococci were first found to be inhibited by condensed phosphate, resulting in a decrease of plaque formation and dental caries (5, 39). The ability of poly(P) to chelate divalent cations is regarded as relevant to the antibacterial effects of poly(P), contributing to cell division i...

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Sequential action of R- and K-specific gingipains of Porphyromonas gingivalis in the generation of the heam-containing pigment from oxyhaemoglobin

Cited by 46 publications

References 21 publications

Bacillus anthracis Overcomes an Amino Acid Auxotrophy by Cleaving Host Serum Proteins

Bacillus anthracis Overcomes an Amino Acid Auxotrophy by Cleaving Host Serum Proteins

Using Tn-seq To Identify Pigmentation-Related Genes of Porphyromonas gingivalis: Characterization of the Role of a Putative Glycosyltransferase

Antibacterial Action of Polyphosphate on Porphyromonas gingivalis

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