A Distinct Type of Pilus from the Human Microbiome.

Pili are proteinaceous polymers of linked pilins that protrude from the cell surface of many bacteria and often mediate adherence and virulence. We investigated a set of 20 Bacteroidia pilins from the human microbiome whose structures and mechanism of assembly were unknown. Crystal structures and biochemical data revealed a diverse protein superfamily with a common Greek-key ß sandwich fold with two transthyretin-like repeats that polymerize into a pilus through a strand-exchange mechanism. The assembly mechanism of the central, structural pilins involves proteinase-assisted removal of their N-terminal ß strand, creating an extended hydrophobic groove that binds the C-terminal donor strands of the incoming pilin. Accessory pilins at the tip and base have unique structural features specific to their location, allowing initiation or termination of the assembly. The Bacteroidia pilus, therefore, has a biogenesis mechanism that is distinct from other known pili and likely represents a different type of bacterial pilus.

ELISA-based highly sensitive assay system for the detection of endogenous NGLY1 activity.

Cytosolic peptide:N-glycanase (NGLY1, PNGase) is an enzyme that cleaves N-glycans from misfolded glycoproteins. In 2012, a human genetic disorder, NGLY1 deficiency, was first reported to be caused by mutations of the NGLY1 gene. Since then, there has been rapid progresses on NGLY1 biology, and gene therapy has been proposed as a promising therapeutic option for NGLY1 deficiency. While a plasma/urine biomarker has also been developed for this disease, detection of NGLY1 activity could be another viable option for early diagnosis of NGLY1 deficiency. Thus far, several in vitro and in cellulo NGLY1 assays have been reported, but those assay systems have several issues that must be addressed in order to develop an assay system compatible for routine clinical examination. Here, we show a facile, highly sensitive in vitro assay system that could be used to detect NGLY1 activity by utilizing its sequence editing function, i.e. conversion of glycosylated Asn into Asp, followed by a detection of newly generated epitope (HA)-tag by anti-HA antibody. Using this ELISA-based assay, we detected endogenous NGLY1 activity in as little as 2 µg of crude extract, which is the equivalent of 5 × 103 cells. Our system also detects NGLY1 activity from cells with compromised NGLY1 activity, such as iPS cells from patient samples. This assay system could be applied in future clinical examinations to achieve an early diagnosis of NGLY1 deficiency.

Incidence of interstitial lung disease in patients with breast cancer: a nationwide database study in Japan.

Aim: This study estimated the incidence of moderate-to-severe drug-induced interstitial lung disease (ILD) among patients with breast cancer in Japan. Methods: We analyzed a large nationwide database of patients with breast cancer treated with anticancer therapies between 2009 and 2022. ILD was identified using diagnostic codes and treatment records. Results: Of the 81,601 patients, 1042 developed ILD requiring corticosteroids, corresponding to an incidence rate of 1.41 per 100 person-years. The incidence varied across years and treatment regimens. Most ILD incidents occurred within the initial 90-day period post-anticancer therapy initiation. Conclusion: Increase in ILD cases and potential risk variations among treatments underline the importance of continued monitoring, especially during treatment onset, and ILD management in patients with breast cancer undergoing therapy.

This article investigates how often a lung condition known as interstitial lung disease (ILD) occurs in patients treated for breast cancer in Japan. ILD can cause inflammation and damage to the lungs and can be a side effect of some cancer treatments. The study looked at over 81,000 patients with breast cancer from 2009 to 2022. A total of 1042 patients developed ILD that required treatment with steroids to reduce inflammation. This number suggests that ILD occurred in 1.41 out of every 100 patients treated each year. The study noted that the chances of developing ILD varied over the years and depended on the type of cancer treatment. The findings showed that ILD is a risk factor for patients undergoing breast cancer treatment, and the risk can change depending on the treatment they receive. This highlights the importance of doctors keeping a close eye on their patients, especially early in the treatment process, to identify and manage any signs of ILD. Careful monitoring can help improve the health and treatment outcomes of patients with breast cancer. The study also points to the need for more research to understand why ILD occurs and how to prevent or treat it.

Development of a Communication Tool between Patients and Physicians for Recognizing COPD Exacerbations in Japan.

In Japan, exacerbations are underreported compared with other countries, possibly due in part to a failure to recognize them. This study aimed to create a simple chronic obstructive pulmonary disease (COPD) Exacerbation Recognition Tool (CERT-J) specifically for Japanese patients. Patients ≥40 years with confirmed COPD or asthma-COPD overlap were included. Focus groups were held to identify words and phrases used by patients to describe symptoms associated with an exacerbation, resulting in candidate items being identified. Following cognitive debriefing, the items were refined based on item frequency, level of endorsement and effect of demographic factors. Exploratory factor analysis (EFA) was then performed to inform an expert panel's choice of items to form the new tool. A total of 41 patients were included in the focus groups and nine patients performed the cognitive debrief. Following this, the expert panel identified 26 items for testing in a further 100 patients (mean age 72 years, forced expiratory volume in 1 s 54.8% predicted and 1.8 exacerbations in the preceding 12 months). Eleven items were associated with breathlessness or activity limitation and seven of these were the most frequently endorsed. EFA identified four factors, with one (breathlessness) being dominant. The expert panel recommended that the CERT-J should include six items: breathlessness and activity limitation (3 items), cough (1 item) and phlegm (2 items). The final CERT-J should benefit patients with COPD by providing them with an increased understanding and recognition of exacerbations.Clinical Trial Registration: GSK K.K (jRCT1080224526).

Rab44 Deficiency Induces Impaired Immune Responses to Nickel Allergy.

Rab44 was recently identified as an atypical Rab GTPase that possesses EF-hand and coiled-coil domains at the N-terminus, and a Rab-GTPase domain at the C-terminus. Rab44 is highly expressed in immune-related cells such as mast cells, macrophages, osteoclasts, and granulocyte-lineage cells in the bone marrow. Therefore, it is speculated that Rab44 is involved in the inflammation and differentiation of immune cells. However, little is known about the role of Rab44 in inflammation. In this study, we showed that Rab44 was upregulated during the early phase of differentiation of M1- and M2-type macrophages. Rab44-deficient mice exhibited impaired tumor necrosis factor alpha and interleukin-10 production after lipopolysaccharide (LPS) stimulation. The number of granulocytes in Rab44-deficient mice was lower, but the lymphocyte count in Rab44-deficient mice was significantly higher than that in wild-type mice after LPS stimulation. Moreover, Rab44-deficient macrophages showed impaired nickel-induced toxicity, and Rab44-deficient mice showed impaired nickel-induced hypersensitivity. Upon nickel hypersensitivity induction, Rab44-deficient mice showed different frequencies of immune cells in the blood and ears. Thus, it is likely that Rab44 is implicated in immune cell differentiation and inflammation, and Rab44 deficiency induces impaired immune responses to nickel allergies.

Insertional Inactivation and Gene Complementation of Prevotella intermedia Type IX Secretion System Reveals Its Indispensable Roles in Black Pigmentation, Hemagglutination, Protease Activity of Interpain A, and Biofilm Formation.

Prevotella intermedia, a Gram-negative oral anaerobic bacterium, is frequently isolated from the periodontal pockets of patients with chronic periodontitis. In recent years, the involvement of the bacterium in respiratory tract infections as well as in oral infections has been revealed. P. intermedia possesses several potent virulence factors, such as cysteine proteinase interpain A encoded by the inpA gene. The genome of P. intermedia carries genes of the type IX secretion system (T9SS), which enables the translocation of virulence factors across the outer membrane in several pathogens belonging to the phylum Bacteroidetes; however, it is still unclear whether the T9SS is functional in this microorganism. Recently, we performed targeted mutagenesis in the strain OMA14 of P. intermedia. Here, we successfully obtained mutants deficient in inpA and the T9SS component genes porK and porT. None of the mutants exhibited protease activity of interpain A. The porK and porT mutants, but not the inpA mutant, showed defects in colony pigmentation, hemagglutination, and biofilm formation. We also obtained a complemented strain for the porK gene that recovered all the above abilities. These results indicate that T9SS functions in P. intermedia and that interpain A is one of the T9SS cargo proteins. IMPORTANCE The virulence factors of periodontal pathogens such as Prevotella intermedia have not been elucidated. Using our established procedure, we succeeded in generating type IX secretion system mutants and gene complementation strains that might transfer virulence factors to the bacterial surface. The generated strains clearly indicate that T9SS in P. intermedia is essential for colonial pigmentation, hemagglutination, and biofilm formation. These results indicated that interpain A is a T9SS cargo protein.

Occurrence of free N-glycans with a single GlcNAc at the reducing termini in animal sera.

Recent studies demonstrated the occurrence of sialyl free N-glycans (FNGs) in sera from a variety of animals. Unlike the intracellular FNGs that mainly carry a single N-acetylglucosamine at their reducing termini (Gn1-type), these extracellular FNGs have an N,N'-diacetylchitobiose at their reducing termini (Gn2-type). The detailed mechanism for how they are formed, however, remains unclarified. In this study, we report on an improved method for isolating FNGs from sera and found that, not only sialyl FNGs, but also neutral FNGs are present in animal sera. Most of the neutral oligomannose-type FNGs were found to be Gn1-type. We also found that a small portion of sialyl FNGs were Gn1-type. The ratio of Gn1-type sialyl FNGs varies between species, and appears to be partially correlated with the distribution of lysosomal chitobiase activity. We also identified small sialylated glycans similar to milk oligosaccharides, such as sialyl lactose or sialyl N-acetyllactosamine in sera. Our results indicate that there are varieties of free oligosaccharides in sera and the mechanism responsible for their formation is more complicated than currently envisaged.

Standards of Conducts for Biostatisticians and Stem Cell Researchers: A Call for Self-formulated Aspirational Ethics Over Built-in Prohibitive Ethics.

We proposed the Standards of Conducts to provide a general framework that will serve as the basis for guiding each biostatistician and stem cell researcher to formulate their personal standards, rather than as rules with which they are required to comply. Given the responsibility and characteristics of their work, they are expected to maintain independence and work autonomously as professionals. Each of the Standards of Conducts comprises a preamble, mission and values to uphold, Standards of Conducts (10 items), and background. When one internalizes "self-formulated" standards, to make excuses for oneself would be akin to a self-betrayal; responsible actions can be anticipated. If one begins and continues to consider "who I am and what do I work for," this will become their inner energy, and a source of motivation and pride to inspire oneself. In addition, this aspirational style might help citizens to recognize the autonomous stance of the professional body and that they share the same values.

The prevalence, burden of disease, and healthcare utilization of patients with eosinophilic granulomatosis with polyangiitis in Japan: a retrospective, descriptive cohort claims database study.

OBJECTIVES: To estimate eosinophilic granulomatosis with polyangiitis (EGPA) prevalence and disease burden in patients with newly diagnosed EGPA in Japan. METHODS: This retrospective descriptive cohort study (GSK ID: 209751, HO-18-19652) used administrative claim data from patients (aged ≤74 years) with EGPA (study period: January 1, 2005-December 31, 2017), identified from their first ICD-10 code for EGPA (index). Data were examined during the 12 months before (baseline) and 12 months following the index date (follow-up). EGPA prevalence, respiratory comorbidities, all-cause healthcare utilization, and oral corticosteroid (OCS) use were assessed. RESULTS: EGPA prevalence (95%CI) increased from 4.2 (0,23.7)/million people (2005) to 38.0 (31.8,45.1)/million people (2017), was generally more common in females versus males, and increased with age. Of the 45 patients with newly diagnosed EGPA, 57.8% had acute bronchitis and 42.2% had upper respiratory tract infections during baseline. During follow-up, 60.0% of patients were hospitalized at least once and 77.8% used OCS (OCS dependent [≥80% of days]: 73.1%). CONCLUSIONS: In Japan, EGPA prevalence increased over time, was generally more common in females, and increased with patient age. EGPA burden was high; respiratory comorbidities were common, and most patients required hospitalization and OCS use. Our data suggest additional EGPA treatment options are needed.

P*R*O*P: a web application to perform phylogenetic analysis considering the effect of gaps.

BACKGROUND: Phylogenetic analysis strongly depends on evolutionary models. Most evolutionary models for estimating genetic differences and phylogenetic relationships do not treat gap sites in the alignment of sequences. Appropriately incorporating evolutionary information of sites containing insertions and deletions into genetic difference measures will be improve the accuracy of phylogenetic estimates. RESULTS: We introduced a new measure for estimating genetic differences, and presented P*R*O*P, a web application for performing phylogenetic analysis based on genetic difference considering the effect of gaps. As an example of phylogenetic analysis using P*R*O*P, we used complete p53 amino acid sequences of 31 organisms and illustrated that the genetic differences with and without information on sites containing gaps result in trees with different topologies. CONCLUSIONS: P*R*O*P is available at https://www.rs.tus.ac.jp/bioinformatics/prop and the user can perform phylogenetic analysis by uploading sequence data on the website. The most distinctive feature of P*R*O*P is its genetic difference that is estimated without eliminating gap sites for alignment sequences, which helps users detect meaningful difference in an evolutionary process. The source code is available in GitHub: https://github.com/TUS-Satolab/PROP .

Bisecting GlcNAc Is a General Suppressor of Terminal Modification of N-glycan.

Glycoproteins are decorated with complex glycans for protein functions. However, regulation mechanisms of complex glycan biosynthesis are largely unclear. Here we found that bisecting GlcNAc, a branching sugar residue in N-glycan, suppresses the biosynthesis of various types of terminal epitopes in N-glycans, including fucose, sialic acid and human natural killer-1. Expression of these epitopes in N-glycan was elevated in mice lacking the biosynthetic enzyme of bisecting GlcNAc, GnT-III, and was conversely suppressed by GnT-III overexpression in cells. Many glycosyltransferases for N-glycan terminals were revealed to prefer a nonbisected N-glycan as a substrate to its bisected counterpart, whereas no up-regulation of their mRNAs was found. This indicates that the elevated expression of the terminal N-glycan epitopes in GnT-III-deficient mice is attributed to the substrate specificity of the biosynthetic enzymes. Molecular dynamics simulations further confirmed that nonbisected glycans were preferentially accepted by those glycosyltransferases. These findings unveil a new regulation mechanism of protein N-glycosylation.

Biofilm Spreading by the Adhesin-Dependent Gliding Motility of Flavobacterium johnsoniae. 1. Internal Structure of the Biofilm.

The Gram-negative bacterium Flavobacterium johnsoniae employs gliding motility to move rapidly over solid surfaces. Gliding involves the movement of the adhesin SprB along the cell surface. F. johnsoniae spreads on nutrient-poor 1% agar-PY2, forming a thin film-like colony. We used electron microscopy and time-lapse fluorescence microscopy to investigate the structure of colonies formed by wild-type (WT) F. johnsoniae and by the sprB mutant (ΔsprB). In both cases, the bacteria were buried in the extracellular polymeric matrix (EPM) covering the top of the colony. In the spreading WT colonies, the EPM included a thick fiber framework and vesicles, revealing the formation of a biofilm, which is probably required for the spreading movement. Specific paths that were followed by bacterial clusters were observed at the leading edge of colonies, and abundant vesicle secretion and subsequent matrix formation were suggested. EPM-free channels were formed in upward biofilm protrusions, probably for cell migration. In the nonspreading ΔsprB colonies, cells were tightly packed in layers and the intercellular space was occupied by less matrix, indicating immature biofilm. This result suggests that SprB is not necessary for biofilm formation. We conclude that F. johnsoniae cells use gliding motility to spread and maturate biofilms.

Biofilm Spreading by the Adhesin-Dependent Gliding Motility of Flavobacterium johnsoniae: 2. Role of Filamentous Extracellular Network and Cell-to-Cell Connections at the Biofilm Surface.

Flavobacterium johnsoniae forms a thin spreading colony on nutrient-poor agar using gliding motility. As reported in the first paper, WT cells in the colony were sparsely embedded in self-produced extracellular polymeric matrix (EPM), while sprB cells were densely packed in immature biofilm with less matrix. The colony surface is critical for antibiotic resistance and cell survival. We have now developed the Grid Stamp-Peel method whereby the colony surface is attached to a TEM grid for negative-staining microscopy. The images showed that the top of the spreading convex WT colonies was covered by EPM with few interspersed cells. Cells exposed near the colony edge made head-to-tail and/or side-to-side contact and sometimes connected via thin filaments. Nonspreading sprB and gldG and gldK colonies had a more uniform upper surface covered by different EPMs including vesicles and filaments. The EPM of sprB, gldG, and WT colonies contained filaments ~2 nm and ~5 nm in diameter; gldK colonies did not include the latter. Every cell near the edge of WT colonies had one or two dark spots, while cells inside WT colonies and cells in SprB-, GldG-, or GldK-deficient colonies did not. Together, our results suggest that the colony surface structure depends on the capability to expand biofilm.

PorM, a core component of bacterial type IX secretion system, forms a dimer with a unique kinked-rod shape.

Porphyromonas gingivalis, which is a major pathogen of the periodontal disease, secrets virulence factors such as gingipain proteases via the type IX secretion system (T9SS). T9SS consists of a trans-periplasmic core complex, the outer membrane translocon complex and the cell-surface complex attached on the outer membrane. PorM is a major component of the trans-periplasmic core complex and is believed to connect the outer membrane component with the inner membrane component. Recent structural studies have revealed that the periplasmic region of GldM, a PorM homolog of a gliding bacterium, consist of four domains and forms a dimer with a straight rod shape. However, only fragment structures are known for PorM. Moreover, one of the PorM fragment structure shows a kink. Here we show the structure of the entire structure of the periplasmic region of PorM (PorMp) at 3.7 Å resolution. PorMp is made up of four domains and forms a unique dimeric structure with an asymmetric, kinked-rod shape. The structure and the following mutational analysis revealed that R204 stabilizes the kink between the D1 and D2 domains and is essential for gingipains secretion, suggesting that the kinked structure of PorM is important for the functional T9SS formation.

Immunoglobulin-like domains of the cargo proteins are essential for protein stability during secretion by the type IX secretion system.

The periodontal pathogen Porphyromonas gingivalis secretes many potent virulence factors using the type IX secretion system (T9SS). T9SS cargo proteins that have been structurally determined by X-ray crystallography are composed of a signal peptide, functional domain(s), an immunoglobulin (Ig)-like domain and a C-terminal domain. Role of the Ig-like domains of cargo proteins in the T9SS has not been elucidated. Gingipain proteases, which are cargo proteins of the T9SS, were degraded when their Ig-like domains were lacking or truncated. The degradation was dependent on the activity of a quality control factor, HtrA protease. Another T9SS cargo protein, HBP35, which has a thioredoxin domain as a functional domain, was analyzed by X-ray crystallography, revealing that HBP35 has an Ig-like domain after the thioredoxin domain and that the hydrophobic regions of the thioredoxin domain and the Ig-like domain face each other. HBP35 with substitution of hydrophobic amino acids in the Ig-like domain was degraded depending on HtrA. These results suggest that the Ig-like domain mediates stability of the cargo proteins in the T9SS.

An Extension of the Kimura Two-Parameter Model to the Natural Evolutionary Process.

Accurate estimates of genetic difference are required for research in evolutionary biology. Here we extend the Kimura two-parameter (K2P) model by considering gaps (insertions and/or deletions) and introduce a new measure for estimating genetic difference between two nucleotide sequences in terms of nucleotide changes that have occurred during the evolutionary process. Using the nuclear ribosomal DNA internal transcribed spacer 2 region from the genus Physalis, we demonstrate that species identification and phylogenetic studies strongly depend on evolutionary models. It is especially noteworthy that the use of different models affects the degree of overlap between intraspecific and interspecific genetic differences. We observe that the percentage of interspecific sequence pairs with values less than the maximum intraspecific genetic difference is 43.2% for the K2P model which is calculated by removing gap sites across all sequences, 22.7% for the K2P model which is calculated by removing gap sites for sequence pairs, and 16.9% for our model which is calculated without removing gap sites. Additionally, the numbers of sequence pairs with interspecific genetic differences of zero are 50 for the K2P model and 29 for our model. The genetic difference measure based on the K2P model, compared to our model, overestimates 21 sequence pairs that are not originally identical. These results indicate the importance of estimating genetic differences under the model of sequence evolution that includes insertions and deletions in addition to substitutions.

Discrimination of colors by red-green color vision-deficient observers through digitally generated red filter.

Previous studies have shown that with the use of tinted lenses (or colored filters), individuals with red-green color vision deficiency (CVD) report an improvement in their performance on certain color vision tests. In this context, this study examines the effects of a digitally generated red-colored filter and identifies the mechanism mainly responsible for the changes in red-green CVD observers' performance on a D-15 arrangement test performed using the filter. We simulate the red filter digitally with the spectral transmittance similar to that of the X-Chrom, which is a red-tinted lens. Fourteen red-green CVD subjects are subjected to the D-15 test on a computer monitor under four filter conditions, consisting of one condition without the filter and three conditions with the filter, corresponding to the opacity of the red filter. The results show that while the simulated red filter improves the performance of deutans to arrange the caps in the D-15 test, this is not the case for protans. In addition, considerations based on the human cone-contrast model enable us to identify that the improvement in deutan observers largely results from the increase in the luminance contrast between stimuli and a background. To summarize, the red filter simulated in this study induces different changes in the red-green CVD observer luminance contrast between the protan and deutan types, with the result that the performance of deuteranopes improves while that of protanopes deteriorates.

The prevalence and disease burden of severe eosinophilic asthma in Japan.

Background: There are limited data on the prevalence and burden of severe eosinophilic asthma (SEA) both in Japan and globally. This study aimed to assess the prevalence and burden of SEA in Japan. Methods: This study was a retrospective, observational cohort analysis using health records or health insurance claims from patients with severe asthma treated at Kyoto University Hospital. The primary outcome was the prevalence of SEA, defined as a baseline blood eosinophil count ≥300 cells/µL. Secondary outcomes included frequency and risk factors of asthma exacerbations, and asthma-related healthcare resource utilization and costs. Results: Overall, 217 patients with severe asthma were included; 160 (74%) had eosinophil assessments. Of these, 97cases (61%), 54cases (34%), and 33cases (21%) had a blood eosinophil count ≥150, ≥300, and ≥500 cells/µL, respectively. Proportion of SEA was 34%. Blood eosinophil count was not associated with a significantly increased frequency of exacerbations. In the eosinophilic group, lower % forced expiratory volume in 1 second and higher fractional exhaled nitric oxide were predictive risk factors, while the existence of exacerbation history was a predictive risk factor for asthma exacerbations in the non-eosinophilic group. Severe asthma management cost was estimated as ¥357,958/patient-year, and asthma exacerbations as ¥26,124/patient-year. Conclusions: Approximately, one-third of patients with severe asthma in Japan have SEA. While risk factors for exacerbations differed between SEA and severe non-eosinophilic asthma, both subgroups were associated with substantial disease and economic burden. From subgroup analysis, blood eosinophil counts could be an important consideration in severe asthma management.

Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System.

The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32-36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component.

Involvement of PorK, a component of the type IX secretion system, in Prevotella melaninogenica pathogenicity.

Prevotella melaninogenica is a gram-negative anaerobic commensal bacterium that resides in the human oral cavity and is isolated as a pathogen of suppurative diseases both inside and outside the mouth. However, little is known about the pathogenic factors of P. melaninogenica. The periodontal pathogens Porphyromonas gingivalis and Tanerella forsythia secrete virulence factors such as protease and bacterial cell surface proteins via a type IX secretion system (T9SS) that are involved in pathogenicity. P. melaninogenica also possesses all known orthologs of T9SS. In this study, a P. melaninogenica GAI 07411 mutant deficient in the orthologue of the T9SS-encoding gene, porK, was constructed. Hemagglutination and biofilm formation were decreased in the porK mutant. Furthermore, following growth on skim milk-containing medium, the diameters of the halos surrounding the porK mutant were smaller than those of the wild-type strain, suggesting a decrease in secretion of proteases outside the bacterium. To investigate this in detail, culture supernatants of wild-type and porK mutant strains were purified and compared by two-dimensional electrophoresis. In the mutant strain, fewer spots were detected, indicating fewer secreted proteins. In infection experiments, the mortality rate of mice inoculated with the porK mutant strain was significantly lower than in the wild-type strain. These results suggest that P. melaninogenica secretes potent virulence factors via the T9SS that contribute to its pathogenic ability.