Paratyphi B. “
“The utility of specific strains of natural algicidal bacteria isolated from shallow wetland sediments was evaluated against several strains of algae with potential immediate or future commercial value. Two strains of bacteria, Pseudomonas pseudoalcaligenes AD6 and Aeromonas hydrophila AD9, were identified and demonstrated to have algicidal activity against the microalgae Neochloris oleoabundans and Dunaliella tertiolecta. These bacteria were further evaluated for the potential to improve lipid extraction using

a mild solvent extraction approach. Aeromonas hydrophila AD9 showed a nearly 12-fold increase in lipid extraction with D. tertiolecta, PD-0332991 nmr while both bacteria showed a sixfold improvement in lipid extraction with N. oleoabundans. “
“Although GlaxoSmithKline is on the way to launch the new vaccine candidate ‘RTS, S’, the search for suitable antimalarial drugs still remains an exceeding challenge because Plasmodium falciparum-mediated malaria is one of the most lethal diseases in the world. Novel innovative ideas are required to identify new potential molecular targets to be able to fight this lethal parasite efficiently. We

used an unconventional bioinformatics approach to analyze the entire genome and proteome of the Pf3D7 strain. Because the oxygen (O-) content is a decisive parameter that determines the function of a protein, we analyzed the entire Pf3D7 proteome based on O-containing amino acid expression. Our data disclose a total of four proteins encoded by chromosome (Chr)-4 and Chr-9 Alisertib that have an outstanding O-controlled character. The identification of the biological significance of

these proteins could eventually lead to new vital drug targets. “
“Division of Crop Protection Central Plantation Crops Research Institute, Kerala, India Conventional and real-time PCR assays were developed for sensitive and specific detection of Phytophthora colocasiae, an oomycete pathogen that causes leaf blight and corm rot of taro. A set of three primer pairs was designed from regions of the RAS-related protein (Ypt1), G protein alpha-subunit (GPA1) and phospho-ribosylanthranilate isomerase (TRP1) genes. In conventional PCR, the lower limit of detection was 50 pg DNA, whereas in real-time PCR, oxyclozanide the detection limit was 12.5 fg for the primer based on Ypt1 gene. The cycle threshold values were linearly correlated with the concentration of the target DNA (range of R2 = 0.911–0.999). All the primer sets were successful in detecting P. colocasie from naturally infected leaves and tubers of taro. Phytophthora colocasiae was detected from artificially infested samples after 18 and 15 h of postinoculation in conventional and real-time PCR assay, respectively. The developed PCR assay proved to be a robust and reliable technique to detect P.

7,8 Indeed, recent CP-690550 clinical trial policy documents stress the contribution that children, young people and families have to make in shaping the future of health care in the UK.9,10 Therefore, although this study in its entirety explored the views of children and young people with T1DM, their parents and health care professionals, the experiences of children, young people and parents are reported here. The main research aims were: To develop a model of care that will deliver the aspirations of the policy document ‘Making every young person with

diabetes matter’.11 To improve the care provision for children and young people with T1DM in England. The research, entitled ‘Join us on our journey’, was a three-year, multi-site study. Nine acute trusts across the Yorkshire and the Humber region were involved and overall 300 participants throughout the region took part. Of Buparlisib ic50 these, 257 comprised children, young people and parents. The research employed a qualitative approach and process-mapping, using talking groups (a term coined by the children and

young people to describe focus groups), was the main methodological component. The rationale behind using a process-mapping approach was to map out the T1DM journey for children and young people who had the condition, which meant establishing what worked well, what worked less well, where the areas of inefficiency were

to be found and how a particular area needed to improve. In the case of diabetes care provision for children and young people, this approach enabled the complete journey, from diagnosis through to transition from paediatric Progesterone to adult services, to be explored. In keeping with the theme, ‘bus stops’ along a ‘diabetes journey’ were used to represent the different stages along the child’s and young person’s diabetes care pathway (see Box 1). The talking groups used the ‘bus stops’ as a basis for generating discussions and all participants were asked three key questions in relation to each ‘bus stop’: What is currently happening? What is missing? What needs to happen? So, as an example, for ‘bus stop’ 3, participants were asked: What currently happens in terms of managing complications? What is missing? What needs to happen? Bus stop 1 Diagnosis and initial management Bus stop 2 Annual assessment of the continuing care plan and monitoring of complications Bus stop 3 Management of complications Bus stop 4 Structured education Bus stop 5 Mental health and emotional well-being Bus stop 6 Support of child and family Bus stop 7 Early years and school setting Bus stop 8 Promoting good health and healthy choices Bus stop 9 Sexual health and pregnancy Bus stop 10 Transition Bus stop 11 Benefits Children and young people aged 6–25 and their parents participated in the research.

[36] Table 2 stratifies some of the more commonly prescribed drugs that can induce photosensitivity

reactions by types of reactions and drug classes.[30-33] Many of the medications listed in Table 2 are frequently prescribed for travelers, such as antimalarials, or frequently included in travel first aid kits, such as analgesics. Travelers taking these medications should be warned of the potential risks of drug-induced photosensitivity reactions and encouraged to apply and to reapply high-SPF (30+) sunscreens whenever sun-exposed. The management of photosensitivity reactions includes the identification and future avoidance of the offending drug, which may require photopatch BMN-673 testing, anti-inflammatory dressings and ointments, and topical and/or systemic corticosteroids.[31-33] reactions Ibuprofen Naproxen Piroxicam Sulfonamides Tetracyclines Trimethoprim Antifungals: Griseofulvin Voriconazole Antimalarials: Chloroquine Quinine Atenolol Sotalol ACEIs: Captopril Enalapril Calcium channel blockers: Verapamil Diuretics: Bumetanide Furosemide Thiazides Miscellaneous: Amiodarone Methyldopa Carbamazepine MLN0128 nmr Valproate Antipsychotics: Phenothiazines Coal tar Psoralens Retinoids Topical antimicrobials Chemotherapeutics: Fluorouracil Methotrexate Vemurafenib

Hypoglycemics: Metformin Sulfonylureas Miscellaneous additives: Furocoumarins reactions Ketoprofen Piroxicam Quinolones Sulfonamides Antifungals: Griseofulvin Quinidine Thiazides Phenothiazines Some topical sunscreen ingredients: Avobenzone Besides fair-skinned persons,

other special populations at increased risks of UV-induced skin cancers include children, organ transplant recipients ASK1 (OTRs), and persons with sun-sensitive genetic skin diseases. Epidemiological evidence now supports the observations that children who have suffered repeated sunburns are more likely to develop CMM as adolescents and adults than children who have never had sunburns.[6, 7, 37] In 2012, Gamble and colleagues used ultraviolet photography to examine the relationships between severity of prior sun exposure damage and phenotypic CMM risk factors in children and demonstrated that degree of sun damage correlated with all known CMM risk factors including non-Hispanic Caucasian race, red hair, blue eyes, increased facial freckling, and greater number of nevi (all p values < 0.001).[6] In 2012, Vranova and colleagues reported the results of a case-control study on the risks of prior sun exposures in childhood on the subsequent incidence of CMMs and found the number of sunburn episodes to be significantly associated with CMMs in adolescents and adults.

They are commonly

used to manufacture fermented milk products and some species are considered probiotics. Many health benefits are associated with their use, including the ability to modulate the immune system (Gill, GSK-3 activity 1998; Salminen et al., 1998) as well as antitumor, antimetastatic properties (Tomita et al., 1994; Matsuzaki et al., 1996). Intraperitoneal administration of Lactobacillus casei induced the production of cytokines such as interferon γ (IFNγ), interleukin-1 (IL-1) and tumor necrosis factor α (TNFα), which could contribute to the inhibition of tumor growth and increased survival of tumor-bearing mice (Matsuzaki, 1998). Several Lactobacillus species stimulate cells of the innate immune system in vitro, namely natural killer cells (Kato Sorafenib research buy et al., 1984; Haller et al., 1999) and macrophages. Stimulation of these cells can induce proinflammatory cytokines such

as TNFα (Haller et al., 1999), IFNγ and IL-12 (Miettinen et al., 1998; Hessle et al., 1999; Kato et al., 1999; Morita et al., 2002), and regulatory cytokines such as IL-10 (Christensen et al., 2002). TNFα directly induces tumor apoptosis and enhances the tumoricidal activity of macrophages (Wang et al., 1996), while IL-12 has potent antitumor and antimetastatic effects against tumors by the stimulation of cytotoxic CD8+ T cells and natural killer cells. IL-12 also enhances the production of Th1 cytokines such as IFNγ. IL-10 plays a regulatory role in allergy (Akbari et al., 2001) and anti-inflammatory responses

(Kuhn et al., 1993). Toll-like Hydroxychloroquine receptors (TLRs) are pattern recognition receptors that recognize molecules that are common to pathogens, but absent in the host. TLR4 is essential for the recognition of lipopolysaccharide, while lipoproteins from gram-positive bacteria are recognized by TLR2 (Takeuchi et al., 1999). Major cell wall components of gram-positive bacteria, such as peptidoglycan and lipoteichoic acid, signal through TLR2 (Schwandner et al., 1999; Matsuguchi et al., 2003) and stimulate cytokine production. The mannose and Fcγ receptors and CD14 are associated with bacterial phagocytosis, which can also result in cytokine production. Unmethylated CpG dinucleotides in the bacterial DNA have stimulatory effects on mammalian immune cells (Lipford et al., 1998). Hemmi et al. (2000) showed that the cellular response to CpG DNA is mediated by TLR9 as TLR9 knockout mice did not respond to CpG DNA and the immune cells from these mice did not produce inflammatory cytokines upon stimulation with CpG DNA. This study aims to evaluate the immunostimulatory properties of three commonly consumed lactobacilli species: L. casei, Lactobacillus rhamnosus and Lactobacillus bulgaricus. Analysis of splenocyte TNFα, IL-12p40 and IL-10 production after stimulation with ‘live’ and lyophilized lactobacilli was performed. The role of TLRs and phagocytosis in the stimulation of cytokine production was also examined.

Results showed a main effect for having a recent STI on infectiousness beliefs; individuals

who had recently been diagnosed with an STI held significantly greater beliefs that an undetectable viral load renders a person noninfectious. The main effect for viral load and the STI by viral load interactions were not significant. Analyses did not indicate any differences between groups in HIV treatment optimism (see Table 3). Results of the multiple logistic regression with all nonredundant and significant factors associated with a recent STI diagnosis are shown in Table 4. The simultaneous model indicated that fewer years of education, more HIV symptoms, use of cannabis and greater HIV infectiousness beliefs were associated with a recent

STI diagnosis over and above the other factors included in the model. Fourteen per cent of men and women living with HIV/AIDS were Fostamatinib cost diagnosed with a new STI in a 6-month period. These rates of incident STIs are similar to those found in other community samples of people living with HIV/AIDS [28]. Having been diagnosed with a recent STI was proportional for men and women and was not associated with income or receiving HIV treatments. Individuals who had been diagnosed with a co-occurring STI were only slightly younger, were less educated, and were using more alcohol and other drugs. Recently contracting an STI was associated with Rapamycin manufacturer poorer health, including having a lower CD4 cell count, experiencing more HIV-related symptoms, and being less likely to have an undetectable viral load. STI coinfection was also associated with being unaware of one’s viral load, a potential indicator of poor engagement with health care. Together, these findings do not support the notion that improved health status accounts for increases in sexual risk

behaviour in people living with HIV/AIDS. The association between believing that Obatoclax Mesylate (GX15-070) one is less infectious when one’s viral load is undetectable and being diagnosed with an STI was significant even after accounting for age, education, substance use, viral load and other health markers. These findings confirm previous research indicating that infectiousness beliefs play a central role in continued HIV transmission risks for some people living with HIV [1]. The current study is the first we are aware of to report an association between infectiousness beliefs and STI coinfection, a circumstance that increases infectiousness regardless of blood serum viral load. Having contracted an STI was not related to higher rates of unprotected sex. Indeed, greater rates of condom use with nonpositive partners were observed among participants who had contracted an STI.

We presumed other causes or etiological agents responsible for respiratory symptoms in this cohort. The previous study had shown Gefitinib price that influenza A virus was only detected in 0.6%,31 8.1%,32 8.6%,17 and 10.2%,29 respectively, of the hajj pilgrims. Other earlier studies also showed that the crude ILI attack rate among vaccinated persons was significantly lower than control group.21,33 But later, another study showed that influenza vaccine appeared to provide some protection against influenza in immunosuppressive conditions and those hajj pilgrims over the

age of 65 but not in the others.34 In the previous study by Meysamie et al. (2006), the rate of respiratory diseases significantly increased from 35% in year 2004 to 70% in 2005 selleckchem with the increment of influenza vaccination coverage.24 In the era of H1N1 pandemic influenza, the ILI cases increased five times more than baseline rate and the pandemic influenza strain took over the seasonal vaccine strain.35 There was no epidemiological evidence of significant protection by seasonal vaccine against pandemic influenza virus infection.36 Although some cross protection of H3N2 was documented

when the subjects are injected by H2N2 vaccine, the protection of H1N1 pandemic influenza 2009 is not expected after vaccination with H1N1 2008 strain because of major different in antigenic site.37 H1N1 pandemic strain vaccination is expected to be the best solution for ILI prevention at the moment. While waiting for the vaccine to appear in the market, infective control measures were implemented, including to hajj pilgrims. Restricting high-risk Muslims from performing hajj this year was one of the options.38 Regular reminders on personal hygiene, avoiding mass crowds as much as possible, reducing unnecessary exertions and taking a lot of water are very important to minimize the problem

with respiratory symptoms. In conclusion, respiratory symptoms were very common among Malaysian hajj pilgrims. The current protective measures are inadequate to give protection. Future research should be aimed at finding other possible interventions which could reduce respiratory infections. As the number of hajj pilgrims increases also each year, these measures ought to be instituted soon. Future studies should also aim at standardization of the terms used and be done in collaboration with researchers from the host nation. This study was funded by Ministry of Higher Education, Malaysia through Universiti Sains Malaysia Hajj Research Cluster. We also would like to acknowledge the Custodian of Two Holyland Hajj Research Center, University of Umm al Qura, Makkah for support with the accommodation and transportation during research in Makkah; Tabung Haji Malaysia for continuous support; and Ms Rohana Che Yusof and Mr Mohd Bazlan Hafidz Mukrim for helping in the data key-in.

In C. burnetii, little is known about the T4BSS regions and the role they play in click here establishing and/or maintaining infection. Coxiella burnetii T4BSS RI contains genes arranged in three linkage groups: (1) icmWCBU1651icmX, (2) icmVdotACBU1647, and (3) icmTicmSdotDdotCdotBCBU1646. We used reverse transcriptase (RT)-PCR to demonstrate transcriptional linkage within the groups, and that icmX, icmV,

and icmT are transcribed de novo by 8 h post infection (hpi). We then examined the transcript levels for icmX, icmW, icmV, dotA, dotB, and icmT during the first 24 h of an infection using quantitative RT-PCR. The expression initially increased for each gene, followed by a decrease at 24 hpi. Subsequently, we analyzed IcmT protein levels during infection and determined that the expression increases significantly from 8 to 24 hpi and then remains relatively constant. These data demonstrate temporal changes

in the RNA of several C. burnetii T4SS RI homologs and the IcmT protein. These changes correspond to early stages of the C. burnetii infectious cycle. Coxiella burnetii is an intracellular pathogen that exhibits a biphasic life cycle that starts with the environmentally stable small-cell variant (SCV) form and converts into the metabolically active and replicative large-cell variant (LCV) form during the first 24 h post infection (hpi) (McCaul & Williams, 1981; McCaul, 1991; Heinzen et al., 1999). Upon infection of a host cell, C. burnetii is trafficked along the endocytic pathway and eventually resides within a parasitophorous vacuole Ivacaftor concentration (PV) retaining the features of a mature

phagolysosome (Akporiaye et al., 1983; Heinzen et al., 1996; Ghigo et al., 2002; Gutierrez et al., 2005; Sauer et al., 2005; Howe & Heinzen, 2006). The early trafficking, enlargement, and maintenance of the C. burnetii PV is dependent Thiamine-diphosphate kinase on C. burnetii protein synthesis (Howe et al., 2003a, b). Infected cells treated with chloramphenicol early during infection contained small tightly bound LAMP-1-positive PVs containing single C. burnetii dispersed throughout the host cell (Howe et al., 2003a, b). However, with the removal of the chloramphenicol, vacuolar fusion resumed, resulting in spacious PVs (SPVs) containing multiple C. burnetii (Howe et al., 2003a, b). Coxiella burnetii-infected cells treated with carbenicillin or nalidixic acid were found to have mature SPVs containing multiple nonreplicating C. burnetii, suggesting that vacuolar development requires metabolically active C. burnetii and is not dependent on bacterial density for complete PV maturation (Howe et al., 2003a, b). These studies demonstrate that the expression of C. burnetii genes during the first 24 hpi of PV niche establishment is crucial for the development of a productive infection.(Coleman et al., 2004). Interestingly, during PV establishment, C.

Positive for oxidase, catalase, nitrate reduction, and hydrolysis of esculin, gelatin, Tween 40, and Tween 80. Negative for indole production, acid production from glucose (fermentation), arginine dihydrolase, urease, β-galactosidase, and hydrolysis of starch. In API ZYM system, cells are positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, and naphthol-AS-BI-phosphohydrolase, but negative for all other enzymes. In API 50 CH tests, acid is produced oxidatively from d-glucose, esculin, and 5-ketogluconate (weakly positive). None of the other

carbon sources were oxidized in the API 50 CH tests. The cells utilize the following compounds as a carbon and energy source by conventional methods: d-glucose, trehalose, acetate, caprate, caproate, propionate, pyruvate, and l-alanine, but not the following compounds: N-acetyl-glucosamine, Y-27632 clinical trial l-arabinose, d-arabitol, d-fructose, d-galactose, d-mannitol, d-mannose, l-rhamnose, d-sorbitol, d-xylose, lactose, cellobiose, maltose, sucrose, glycerol, myo-inositol, adipate, citrate, formate, gluconate, lactate, dl-malate, succinate, l-asparagine, l-asparate, l-glutamate, l-histidine, l-leucine, l-serine,

click here l-threonine, l-phenylalanine, l-proline, benzoate, and 4-hydroxybenzoate. The DNA G + C content is 48.6 mol%. The type strain, KU41ET (=JCM 17778T), was isolated from seawater obtained from the coastal region of Ishigaki Island, Japan. We are grateful to Professor Hans 3-oxoacyl-(acyl-carrier-protein) reductase G. Trüper for his help with the genus and species name. This work was supported by ‘Strategic Project to Support

the Formation of Research Bases at Private Universities’: Matching Fund Subsidy from MEXT (Ministry of Education, Culture, Sports, Science and Technology), 2008–2012. “
“Bioinformatic and electron microscopy analyses indicate that the composition of the B. megaterium QM B1551 spore coat is likely to differ substantially from other Bacillus species. We report here on the identification and characterisation of novel B. megaterium proteins that appear to be abundant in the spore coat. All three proteins, encoded by loci BMQ_0737, BMQ_3035 and BMQ_4051, were identified by proteomic analysis of alkaline detergent extracts from mature spores. Putative spore coat proteins were characterised by transcriptional, reporter-fusion and mutagenesis analyses supported by fluorescence and transmission electron microscopy. These analyses revealed that BMQ_0737 is a novel morphogenetic protein that is required for the correct assembly of the B. megaterium outer spore coat and exosporium, both of which are structurally compromised or missing in BMQ_0737 null mutant spores. “
“Upon infection of the gastric epithelial cells, the Helicobacter pylori cytotoxin-associated gene A (CagA) virulence protein is injected into the epithelial cells via the type IV secretion system (TFSS), which is dependent on cholesterol.

Positive for oxidase, catalase, nitrate reduction, and hydrolysis of esculin, gelatin, Tween 40, and Tween 80. Negative for indole production, acid production from glucose (fermentation), arginine dihydrolase, urease, β-galactosidase, and hydrolysis of starch. In API ZYM system, cells are positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, and naphthol-AS-BI-phosphohydrolase, but negative for all other enzymes. In API 50 CH tests, acid is produced oxidatively from d-glucose, esculin, and 5-ketogluconate (weakly positive). None of the other

carbon sources were oxidized in the API 50 CH tests. The cells utilize the following compounds as a carbon and energy source by conventional methods: d-glucose, trehalose, acetate, caprate, caproate, propionate, pyruvate, and l-alanine, but not the following compounds: N-acetyl-glucosamine, R428 l-arabinose, d-arabitol, d-fructose, d-galactose, d-mannitol, d-mannose, l-rhamnose, d-sorbitol, d-xylose, lactose, cellobiose, maltose, sucrose, glycerol, myo-inositol, adipate, citrate, formate, gluconate, lactate, dl-malate, succinate, l-asparagine, l-asparate, l-glutamate, l-histidine, l-leucine, l-serine,

Vincristine l-threonine, l-phenylalanine, l-proline, benzoate, and 4-hydroxybenzoate. The DNA G + C content is 48.6 mol%. The type strain, KU41ET (=JCM 17778T), was isolated from seawater obtained from the coastal region of Ishigaki Island, Japan. We are grateful to Professor Hans Flavopiridol (Alvocidib) G. Trüper for his help with the genus and species name. This work was supported by ‘Strategic Project to Support

the Formation of Research Bases at Private Universities’: Matching Fund Subsidy from MEXT (Ministry of Education, Culture, Sports, Science and Technology), 2008–2012. “
“Bioinformatic and electron microscopy analyses indicate that the composition of the B. megaterium QM B1551 spore coat is likely to differ substantially from other Bacillus species. We report here on the identification and characterisation of novel B. megaterium proteins that appear to be abundant in the spore coat. All three proteins, encoded by loci BMQ_0737, BMQ_3035 and BMQ_4051, were identified by proteomic analysis of alkaline detergent extracts from mature spores. Putative spore coat proteins were characterised by transcriptional, reporter-fusion and mutagenesis analyses supported by fluorescence and transmission electron microscopy. These analyses revealed that BMQ_0737 is a novel morphogenetic protein that is required for the correct assembly of the B. megaterium outer spore coat and exosporium, both of which are structurally compromised or missing in BMQ_0737 null mutant spores. “
“Upon infection of the gastric epithelial cells, the Helicobacter pylori cytotoxin-associated gene A (CagA) virulence protein is injected into the epithelial cells via the type IV secretion system (TFSS), which is dependent on cholesterol.

For immunoblot analysis, HRP-conjugated goat anti-rabbit immunoglobulin G (IgG) (Bio-Rad) was used as the secondary antibody. pKS9 was digested with NcoI (in the sov) and KpnI (in a vector), blunted with T4 DNA polymerase, and ligated to create pKS20. A 0.6-kbp 3′-terminal region of sov was amplified from pKS9 by PCR with 5′-ATGGTACCTATCTCGAGATGTCGTAGTCCGCACTG-3′ (italics: KpnI and XhoI sites) and 5′-CAGGAAACAGCTATGACC-3′. The PCR product was digested with EcoRI and KpnI and cloned into a 5.6-kbp EcoRI–KpnI-digested

fragment from pKS9 to create pKS21. Similarly, a 0.65-kbp 3′-terminal region of the sov fragment was amplified with 5′-ATGGTACCTAGCTAGCTGAGCTGACAAGCGGATGG-3′ (italics: KpnI and NheI sites) and 5′-CAGGAAACAGCTATGACC-3′; then, the PCR product was digested with Selleckchem NVP-BEZ235 EcoRI and KpnI and cloned into a 5.6-kbp EcoRI–KpnI-digested fragment from pKS9 to create pKS22. pKS24 was constructed by ligation of a 6.2-kbp NheI–KpnI-digested fragment from pKS22 and

an annealed-oligonucleotide linker, 5′-CTAGCTTCCCTATCACGAATTCGAATTTCGGCGTCAGCTAGGTAC-3′/5′-CTAGCTGACGCCGAAATTCGAATTCGTGATAGGGAAG-3′ (italics: BstBI site). pKS24 was digested with BstBI, blunted with T4 DNA polymerase, and ligated to construct pKS23. pKS24 was digested with BstBI and KpnI and ligated with an annealed-oligonucleotide linker [5′-CGAATTTCGGCGTGAGCTCGAGGTAC-3′/5′-CTCGAGCTCACGCCGAAATT-3′ (italics: SacI site)] to create pKS25, which contains a SacI site. pKS26–pKS31 were constructed by ligation Opaganib price of a 6.2-kbp SacI–KpnI-digested fragment from pKS25 with the following annealed-oligonucleotide linkers: 5′-TCTAGATATCAGATCTGGTAC-3′/5′-CAGATCTGATATCTAGAAGCT-3′ ROS1 (for pKS26), 5′-TCCGTTGATATCAGATCTGGTAC-3′/5′-CAGATCTGATATCAACGGAAGCT-3′ (for pKS27), 5′-TCCGTTTCTGATATCAGATCTGGTAC-3′/5′-CAGATCTGATATCAGAAACGGAAGCT-3′

(for pKS28), 5′-TCCGTTTCAATTGATATCAGATCTGGTAC-3′/5′-CAGATCTGATATCAATTGAAACGGAAGCT-3′ (for pKS29), 5′-TCCGTTTCAATCTGTGATATCAGATCTGGTAC-3′/5′-CAGATCTGATATCACAGATTGAAACGGAAGCT-3′ (for pKS30), and 5′-TCCGTTTCAATCTGACGTGATATCAGATCTGGTAC-3′/5′-CAGATCTGATATCACGTCAGATTGAAACGGAAGCT-3′ (for pKS31). pKS20, pKS21, pKS22, pKS23, pKS24, pKS26, pKS27, pKS28, pKS29, pKS30, and pKS31 were linearized and used to construct P. gingivalis mutants 83K14, 83K15, 83K16, 83K17, 83K18, 83K19, 83K20, 83K21, 83K22, 83K23, and 83K24, respectively, by electroporation. Deletion mutations of 83K14–24 were confirmed similarly as described above. A P. gingivalis cell culture was centrifuged. The cell pellets were washed, suspended in PBS, and sonicated (with tip #3) to generate the cell extract fraction. The culture supernatant was collected as the extracellular fraction. To determine the expression of gingipains, 3 mL of supernatant was concentrated on an ultrafiltration membrane (10 000 MWCO), diluted with 8 M urea, and concentrated to 0.1 mL. Rgp activity was determined in Tris-HCl (100 mM, pH 8.