A phosphopantetheinyl transferase gene restricted to Porphyromonas.

The phosphopantetheinyl transferases (PPTases) catalyze the post-translational modification of carrier proteins (CPs) from fatty acid synthases (FASs) in primary metabolism and from polyketide synthases (PKSs) and non-ribosomal polypeptide synthases (NRPSs) in secondary metabolism. Based on the conserved sequence motifs and substrate specificities, two types (AcpS-type and Sfp-type) of PPTases have been identified in prokaryotes. We present here that Porphyromonas gingivalis, the keystone pathogen in chronic periodontitis, harbors merely one PPTase, namely PptP. Complementation and gene deletion experiments clearly show that PptP can replace the function of Escherichia coli AcpS and is essential for the growth of P. gingivalis. Purified PptP transfers the 4-phosphopantetheine moiety of CoA to inactive apo-acyl carrier protein (ACP) to form holo-ACP, which functions as an active carrier of the acyl intermediates of fatty acid synthesis. Moreover, PptP exhibits broad substrate specificity, modifying all ACP substrates tested and catalyzing the transfer of coenzyme A (CoA) derivatives. The lack of sequence alignment with known PPTases together with phylogenetic analyses revealed PptP as a new class of PPTases. Identification of the new PPTase gene pptP exclusive in Porphyromonas species reveals a potential target for treating P. gingivalis infections.

Gut bacteria identified in colorectal cancer patients promote tumourigenesis via butyrate secretion.

Emerging evidence is revealing that alterations in gut microbiota are associated with colorectal cancer (CRC). However, very little is currently known about whether and how gut microbiota alterations are causally associated with CRC development. Here we show that 12 faecal bacterial taxa are enriched in CRC patients in two independent cohort studies. Among them, 2 Porphyromonas species are capable of inducing cellular senescence, an oncogenic stress response, through the secretion of the bacterial metabolite, butyrate. Notably, the invasion of these bacteria is observed in the CRC tissues, coinciding with the elevation of butyrate levels and signs of senescence-associated inflammatory phenotypes. Moreover, although the administration of these bacteria into ApcΔ14/+ mice accelerate the onset of colorectal tumours, this is not the case when bacterial butyrate-synthesis genes are disrupted. These results suggest a causal relationship between Porphyromonas species overgrowth and colorectal tumourigenesis which may be due to butyrate-induced senescence.

Gut Microbiota Serves a Predictable Outcome of Short-Term Low-Carbohydrate Diet (LCD) Intervention for Patients with Obesity.

To date, much progress has been made in dietary therapy for obese patients. A low-carbohydrate diet (LCD) has reached a revival in its clinical use during the past decade with undefined mechanisms and debatable efficacy. The gut microbiota has been suggested to promote energy harvesting. Here, we propose that the gut microbiota contributes to the inconsistent outcome under an LCD. To test this hypothesis, patients with obesity or patients who were overweight were randomly assigned to a normal diet (ND) or an LCD group with ad libitum energy intake for 12 weeks. Using matched sampling, the microbiome profile at baseline and end stage was examined. The relative abundance of butyrate-producing bacteria, including Porphyromonadaceae Parabacteroides and Ruminococcaceae Oscillospira, was markedly increased after LCD intervention for 12 weeks. Moreover, within the LCD group, participants with a higher relative abundance of Bacteroidaceae Bacteroides at baseline exhibited a better response to LCD intervention and achieved greater weight loss outcomes. Nevertheless, the adoption of an artificial neural network (ANN)-based prediction model greatly surpasses a general linear model in predicting weight loss outcomes after LCD intervention. Therefore, the gut microbiota served as a positive outcome predictor and has the potential to predict weight loss outcomes after short-term LCD intervention. Gut microbiota may help to guide the clinical application of short-term LCD intervention to develop effective weight loss strategies. (This study has been registered at the China Clinical Trial Registry under approval no. ChiCTR1800015156). IMPORTANCE Obesity and its related complications pose a serious threat to human health. Short-term low-carbohydrate diet (LCD) intervention without calorie restriction has a significant weight loss effect for overweight/obese people. Furthermore, the relative abundance of Bacteroidaceae Bacteroides is a positive outcome predictor of individual weight loss after short-term LCD intervention. Moreover, leveraging on these distinct gut microbial structures at baseline, we have established a prediction model based on the artificial neural network (ANN) algorithm that could be used to estimate weight loss potential before each clinical trial (with Chinese patent number 2021104655623). This will help to guide the clinical application of short-term LCD intervention to improve weight loss strategies.

Revisiting Metchnikoff: Age-related alterations in microbiota-gut-brain axis in the mouse.

Over the last decade, there has been increased interest in the role of the gut microbiome in health including brain health. This is by no means a new theory; Elie Metchnikoff proposed over a century ago that targeting the gut by consuming lactic acid bacteria such as those in yogurt, could improve or delay the onset of cognitive decline associated with ageing. However, there is limited information characterising the relationship between the behavioural and physiological sequelae of ageing and alterations in the gut microbiome. To this end, we assessed the behavioural, physiological and caecal microbiota profile of aged male mice. Older mice (20-21months old) exhibited deficits in spatial memory and increases in anxiety-like behaviours compared to younger mice (2-3months old). They also exhibited increased gut permeability, which was directly correlated with elevations in peripheral pro-inflammatory cytokines. Furthermore, stress exacerbated the gut permeability of aged mice. Examination of the caecal microbiota revealed significant increases in phylum TM7, family Porphyromonadaceae and genus Odoribacter of aged mice. This represents a shift of aged microbiota towards a profile previously associated with inflammatory disease, particularly gastrointestinal and liver disorders. Furthermore, Porphyromonadaceae, which has also been associated with cognitive decline and affective disorders, was directly correlated with anxiety-like behaviour in aged mice. These changes suggest that changes in the gut microbiota and associated increases in gut permeability and peripheral inflammation may be important mediators of the impairments in behavioural, affective and cognitive functions seen in ageing.

Meningitis in adolescents: the role of commensal microbiota.

The pathogen Neisseria meningitidis causes disease amongst infants and adolescents/young adults. Here we argue that disease amongst adolescents is due largely to interaction between N. meningitidis and other members of the upper respiratory tract microbiota, through a metabolic interaction involving exchange of propionic acid.

Porphyromonas gulae 41-kDa fimbriae induced osteoclast differentiation and cytokine production.

Porphyromonas gulae is considered to be associated with canine periodontitis. We have previously reported that the P. gulae American Type Culture Collection (ATCC) 51700 comprised 41-kDa fimbriae. The purpose of the present study was to demonstrate the roles of 41-kDa fimbrial protein in periodontal disease. In this study, we examined the involvement of the 41-kDa fimbrial protein in osteoclast differentiation and cytokine production in murine macrophages. Furthermore, alveolar bone resorption induced by P. gulae infection in rats was evaluated. To estimate osteoclast differentiation, bone marrow cells and MC3T3-G2/PA6 cells were cultured with or without the 41-kDa fimbrial protein for 7 days. BALB/c mouse peritoneal macrophages were stimulated with the 41-kDa fimbrial protein, and the levels of interleukin (IL)-1ß and tumor necrosis factor (TNF)-α production were determined by enzyme-linked immunosorbent assay. Osteoclast differentiation was significantly enhanced by treatment with the 41-kDa fimbrial protein in a dose-dependent manner. The total area of pits formed on the dentine slices with osteoclasts incubated with the 41-kDa fimbrial protein was significantly greater than that of the control. The purified 41-kDa fimbrial protein induced IL-1ß and TNF-α production in BALB/c mouse peritoneal macrophages after 6 hr of incubation in a dose-dependent manner. The bone loss level in rats infected with P. gulae was significantly higher than that of the sham-infected rats. These results suggest that P. gulae 41-kDa fimbriae play important roles in the pathogenesis of periodontal disease.

A large genomic island allows Neisseria meningitidis to utilize propionic acid, with implications for colonization of the human nasopharynx.

Neisseria meningitidis is an important human pathogen that is capable of killing within hours of infection. Its normal habitat is the nasopharynx of adult humans. Here we identify a genomic island (the prp gene cluster) in N. meningitidis that enables this species to utilize propionic acid as a supplementary carbon source during growth, particularly under nutrient poor growth conditions. The prp gene cluster encodes enzymes for a methylcitrate cycle. Novel aspects of the methylcitrate cycle in N. meningitidis include a propionate kinase which was purified and characterized, and a putative propionate transporter. This genomic island is absent from the close relative of N. meningitidis, the commensal Neisseria lactamica, which chiefly colonizes infants not adults. We reason that the possession of the prp genes provides a metabolic advantage to N. meningitidis in the adult oral cavity, which is rich in propionic acid-generating bacteria. Data from classical microbiological and sequence-based microbiome studies provide several lines of supporting evidence that N. meningitidis colonization is correlated with propionic acid generating bacteria, with a strong correlation between prp-containing Neisseria and propionic acid generating bacteria from the genus Porphyromonas, and that this may explain adolescent/adult colonization by N. meningitidis.

Tannerella forsythia BspA increases the risk factors for atherosclerosis in ApoE(-/-) mice.

OBJECTIVE: The aim of this study was to evaluate the effects of Tannerella forsythia and its major surface virulence factor, BspA, on the progression of atherosclerosis in ApoE(-/-) mice and the expression of lipid metabolism-related genes. METHODS: PMA-differentiated THP-1 cells were treated with BspA to detect foam cell formation. The proximal aortas of ApoE(-/-) mice injected with T. forsythia or BspA were stained with oil red O to examine lipid deposition. The serum levels of CRP, HDL, and LDL were detected by ELISA. The liver tissue of T. forsythia- or BspA-injected ApoE(-/-) mice was examined for mRNA expression of lipid metabolism-related genes, such as liver X receptors (LXRα and LXRß) and ATP-binding cassette transporter A1 (ABCA1). RESULTS: Tannerella forsythia and BspA induced foam cell formation in THP-1 cells and accelerated the progression of atherosclerotic lesions in ApoE(-/-) mice. Mouse serum levels of CRP and LDL were increased, and HDL was decreased by T. forsythia and BspA. The expression levels of LXRα and LXRß, and ABCA1 in liver tissue were decreased by T. forsythia and BspA. CONCLUSIONS: Tannerella forsythia and BspA augmented atherosclerotic lesion progression in ApoE(-/-) mice. This process may be associated with downregulation of lipid metabolism-related gene expression.

Occurrence and antimicrobial susceptibility of Porphyromonas spp. and Fusobacterium spp. in dogs with and without periodontitis.

The occurrence of Porphyromonas gulae, Porphyromonas macacae, Fusobacterium nucleatum and Fusobacterium canifelinum in subgingival plaque from dogs with and without periodontitis as well as their antimicrobial susceptibility were evaluated. From 50 dogs with periodontitis were identified 38 P. gulae, 8 P. macacae, 26 F. nucleatum and 15 F. canifelinum, and from 50 dogs without periodontitis were identified 15 P. gulae, 12 F. nucleatum and 11 F. canifelinum. All strains were susceptible to most of the antibiotics tested, however, different resistance rates to clarithromycin, erythromycin and metronidazole among strains were observed. The role of P. gulae, P. macacae, F. nucleatum and F. canifelinum in periodontal disease of household pets needs to be defined to a better prevention and treatment of the canine periodontitis.

Diversity of fimbrillin among Porphyromonas gulae clinical isolates from Japanese dogs.

Porphyromonas gulae, a gram-negative black-pigmented anaerobe, is a pathogen for periodontitis in dogs. An approximately 41-kDa fimbrial subunit protein (FimA) encoded by fimA is regarded as associated with periodontitis. In the present study, the fimA genes of 17 P. gulae strains were sequenced, and classified into two major types. The generation of phylogenetic trees based on the deduced amino acid sequence of FimA of P. gulae strains along with sequences from several strains of Porphyromonas gingivalis, a major cause of human periodontitis, revealed that the two types of FimA (types A and B) of P. gulae were similar to type I FimA and types II and III FimA of P. gingivalis, respectively. A PCR system for classification was established based on differences in the nucleotide sequences of the fimA genes. Analysis of 115 P. gulae-positive oral swab specimens from dogs revealed that 42.6%, 22.6%, and 26.1% of them contained type A, type B, and both type A and B fimA genes, respectively. Experiments with a mouse abscess model demonstrated that the strains with type B fimA caused significantly greater systemic inflammation than those with type A. These results suggest that the FimA proteins of P. gulae are diverse with two major types and that strains with type B fimA could be more virulent.

Porphyromonas somerae sp. nov., a pathogen isolated from humans and distinct from porphyromonas levii.

Porphyromonas levii is an anaerobic, pigmented gram-negative bacillus originally isolated from bovine rumen. We describe 58 human clinical strains of P. levii-like organisms, isolated from various human clinical specimens that are phenotypically similar to the type strain of P. levii, a rumen isolate (ATCC 29147). Our biochemical, comparative 16S rRNA sequence analyses, and DNAlpha-DNA relatedness studies indicate that the human P. levii-like organisms are similar to each other but genetically different from the P. levii type strain isolated from bovine rumen. We therefore propose the name Porphyromonas somerae to encompass the human P. levii-like organisms. P. somerae was predominantly isolated from patients with chronic skin and soft tissue or bone infections, especially in the lower extremities.

Evaluation of a monovalent companion animal periodontal disease vaccine in an experimental mouse periodontitis model.

Periodontal disease in companion animals is clinically similar to that of human periodontal disease. Despite the usage of veterinary procedures and antibiotic therapy, the disease still remains as one of the most highly prevalent disorders seen by veterinarians. The goal of this study was to evaluate the immunogenic properties and vaccine performance of a monovalent canine periodontal disease vaccine in the mouse oral challenge model of periodontitis. Mice vaccinated subcutaneously with inactivated, whole-cell bacterin preparations of Porphyromonas gulae displayed both high titers of anti-P. gulae specific antibodies and significantly reduced alveolar bone loss in response to homologous, heterologous, and cross-species challenge. Based on the results of these studies, a periodontal disease vaccine may be a useful tool in preventing the progression of periodontitis in animals.

Porphyromonas uenonis sp. nov., a pathogen for humans distinct from P. asaccharolytica and P. endodontalis.

Three Porphyromonas species (Porphyromonas asaccharolytica, P. endodontalis, and the novel species that is the subject of the present report, P. uenonis) are very much alike in terms of biochemical characteristics, such as enzyme profiles and cellular fatty acid contents. P. asaccharolytica is distinguished from the other two species by virtue of production of alpha-fucosidase and glyoxylic acid positivity. The novel species is difficult to differentiate from P. endodontalis phenotypically and was designated a P. endodontalis-like organism for some time. However, P. endodontalis is recovered almost exclusively from oral sources and also grows poorly on Biolog Universal Agar, both characteristics that are in contrast to those of the other two organisms. Furthermore, P. uenonis is glycerol positive in the Biolog AN Microplate system. Both P. asaccharolytica and P. uenonis are positive by 13 other tests in the Biolog system, whereas P. endodontalis is negative by all of these tests. P. asaccharolytica grew well in both solid and liquid media without supplementation with 5% horse serum, whereas the other two species grew poorly without supplementation. Sequencing of 16S rRNA revealed about 10% divergence between the novel species and P. endodontalis but less than 2% sequence difference between the novel species and P. asaccharolytica. Subsequent DNA-DNA hybridization studies documented that the novel organism was indeed distinct from P. asaccharolytica. We propose the name Porphyromonas uenonis for the novel species. We have recovered P. uenonis from four clinical infections in adults, all likely of intestinal origin, and from the feces of six children.

Degradation of cross-linked and non-cross-linked arabinoxylans by the intestinal microbiota in children.

In humans, nonstarch polysaccharides (NSP), such as arabinoxylans (AX), are not digested in the upper gut and provide fermentable carbon sources for bacteria growing in the large bowel. Despite the ubiquity of AX in nature, the microbiologic and physiologic consequences of AX digestion in the gut are poorly understood. In this study, we investigated the breakdown of ferulic acid-cross-linked AX (AXF) and non-cross-linked AX in children's intestinal microbiotas, using starch as a readily fermentable polysaccharide for comparative purposes. The experiments were performed using pH-controlled fermentation vessels under anaerobic conditions. The results demonstrated that there was variation in the metabolism of these polysaccharides by colonic microbiotas. AX was always degraded more slowly than starch, while ferulic acid cross-linking reduced the rate of AX fermentation, as shown by fermentation product measurements. Starch digestion was associated with significant acetate and butyrate production, whereas AX breakdown resulted in increased propionate formation. In general, the presence of fermentable carbohydrate significantly increased the total anaerobe counts and eubacterial rRNA concentrations (P < 0.01), while non-cross-linked AX digestion was principally associated with increased viable counts of Bacteroides fragilis group organisms, which was supported by increases in Bacteroides-Porphyromonas-Prevotella group rRNA (P < 0.01). Starch was considerably more bifidogenic than AX in these fermentations. In conclusion, in this study we found that the effects of AX and AXF on the microbial ecology and metabolism of intestinal microbiotas are similar in children and adults.

Outer-membrane pore-forming proteins in gram-negative anaerobic bacteria.

The outer-membrane proteins (OMPs) of bacteria function as the dynamic interface between the bacterium and its surroundings and are involved in maintenance of cell structure, binding a variety of substances, adhesion to other cells, and regulation of transport of both nutrients and bactericidal agents. There is a vast amount of information about aerobic OMPs and their roles in immunogenicity, virulence, and antimicrobial resistance. Knowledge about OMPs in anaerobic bacteria is much sparser. Genetic data present in data banks regarding aerobic porins are not readily helpful in identifying or analyzing anaerobic porins because of the large phylogenetic distance between the aerobic and anaerobic organisms. We recently identified and sequenced the genes for both a porin protein complex and an OmpA protein in Bacteroides fragilis, and the data are summarized here. Also, recent information is presented about similar OMPs found in other gram-negative anaerobic bacteria, including Bacteroides thetaiotaomicron, Bacteroides distasonis, Porphyromonas, and Fusobacterium.

Porphyromonas endodontalis binds, reduces and grows on human hemoglobin.

Porphyromonas endodontalis is a black-pigmented, obligate anaerobic rod-shaped bacterium implicated as playing a major role in endodontic infections. We have previously shown that P. endodontalis requires the porphyrin nucleus, preferably supplied as hemoglobin, as a growth supplement. The bacteria also actively transport free iron, although this activity does not support growth in the absence of a porphyrin source. The purpose of this study was to further investigate the binding and subsequent utilization of human hemoglobin by P. endodontalis. P. endodontalis binds hemoglobin and reduces the Fe(III) porphyrin, resulting in a steady accumulation of ferrous hemoglobin. Reduction of methemoglobin was similar to the extracellular reduction of nitrobluetetrazolium in the presence of oxidizable substrate. Turbidimetric and viable cell determinations showed that P. endodontalis grew when supplied only hemoglobin. Therefore, we conclude that hemoglobin appears to serve as a sole carbon and nitrogen source, and that these bacteria reduce extracellular compounds at the expense of oxidized substrates.

Periodontal pathogens produce quorum sensing signal molecules.

Different species of bacteria important in the composition of dental plaque were tested for production of extracellular autoinducer-like activities that stimulate the expression of the luminescence genes in Vibrio harveyi. Several strains of Prevotella intermedia, Fusobacterium nucleatum, and Porphyromonas gingivalis were found to produce such activities. Interestingly, these bacteria belong to the same phylogenetic group, and they are periodontal pathogens important in the development of periodontal disease. They specifically produce extracellular signaling molecule related autoinducer-2 from V. haveyi. Nevertheless, they seem to be unable to produce homologues of acyl-homoserine lactones. Furthermore, Escherichia coli DH5alpha can be complemented by the introduction of a P. gingivalis gene with high homology to the luxS gene, which has been called luxS(P.g.).

Microbiology of infected poison ivy dermatitis.

We report the aerobic and anaerobic microbiology of secondarily infected poison ivy dermatitis. The study involved retrospective review of clinical and microbiology laboratory records of patients with secondarily infected poison ivy lesions. Bacterial growth was noted in 33 specimens. Aerobic or facultative anaerobic bacteria only were present in 18 (55%) patients, anaerobic bacteria only in seven (21%), and mixed anaerobic-aerobic bacteria in eight (24%). Forty-five isolates were recovered (1.4 per specimen): 27 aerobic or facultative anaerobic bacteria, and 18 strict anaerobes. The predominant aerobic and facultative anaerobic bacteria were Staphylococcus aureus (13 isolates) and group A beta-haemolytic streptococci (six). The predominant anaerobes were Peptostreptococcus spp. (seven isolates), pigmented Prevotella and Porphyromonas spp. (four) and Fusobacterium spp. (two). Single bacterial isolates were recovered in 18 (55%) patients, eight of which were S. aureus. Nineteen of the organisms isolated from 16 (48%) patients produced the enzyme beta-lactamase. Organisms that resided in the mucous membranes close to the lesions predominated in those infections. Enteric gram-negative rods and Bacteroides fragilis group predominated in leg and buttock lesions. Group A beta-haemolytic streptococci, pigmented Prevotella and Porphyromonas and Fusobacterium spp. were most frequently recovered from lesions of the finger, face and neck. The polymicrobial aetiology of secondarily infected poison ivy lesions, and the association of bacterial flora with the anatomical site of the lesions, are demonstrated.

Growth stimulation of Porphyromonas endodontalis by hemoglobin and protoporphyrin IX.

Porphyromonas endodontalis, like other Porphyromonas species, has a complex set of nutritional requirements. In addition to being an obligate anaerobe, the bacterium must be grown in a complex medium consisting of amino acids, reducing agents and heme compounds. P. endodontalis accumulates high concentrations of heme pigments to the extent that colonies appear black on blood agar. This accumulation of heme and the need for these compounds has been characterized as iron requirements by these species. However, in our studies, P. endodontalis demonstrated growth dependence on hemoglobin or protoporphyrin IX but not on free iron. Iron added to other heme compounds actually decreased growth stimulation by porphyrin-containing compounds. P. endodontalis actively transported free iron, but this process did not appear to be critical for growth. The maximum stimulation of growth by protoporphyrin IX, under conditions of iron deprivation, suggests that P. endodontalis requires the porphyrin moiety as a growth factor.

Experimental root canal infections in conventional and germ-free mice.

A small animal model was evaluated to study the interrelationships between microorganisms after their implantation in root canals (inferior central incisors) using germ-free (GF) and conventional (CV) mice. The selected microorganisms were: Porphyromonas endodontalis (ATCC 35406), Eubacterium lentum (ATCC 25559), Peptostreptococcus anaerobius (ATCC 27337), Fusobacterium nucleatum (ATCC 10953), Escherichia coli (ATCC 25922), and Enterococcus faecalis (ATCC 4083). Only P. anaerobius, E. coli, and E. faecalis, respectively, were able to colonize when inoculated alone into the root canal of both CV and GF mice. E. lentum, when inoculated alone colonized only in CV animals. P. endodontalis and F. nucleatum were unable to colonize in CV and GF animals after single inoculation. It is concluded that the experimental animal model presented herein is valuable for ecological studies of root canal infections and that only some strict anaerobic bacteria are able to colonize mice root canals when inoculated by themselves alone in pure culture.