Although the incidence of gastric cancer in the Dominican Republic is not high, the disease remains a significant health problem. We first conducted a detailed analysis of Helicobacter pylori status in the Dominican Republic. In total, 158 patients (103 females and 55 males; mean age 47.1±16.2 years) were recruited. The status of H. pylori infection was determined based on four tests: rapid urease test, culture test, histological test and immunohistochemistry. The status of cagA and vacA genotypes in H. pylori was examined using PCR and gene sequencing. The overall prevalence of H. pylori infection was 58.9 %. No relationship was found between the H. pylori infection rate and the age range of 17–91 years. Even in the youngest group (patients aged <29 years), the H. pylori infection rate was 62.5 %. Peptic ulcer was found in 23 patients and gastric cancer was found in one patient. The H. pylori infection rate in patients with peptic ulcer was significantly higher than that in patients with gastritis (82.6 versus 54.5 %, P<0.01). The cagA-positive/vacA s1m1 genotype was the most prevalent (43/64, 67.2 %). Compared with H. pylori-negative patients, H. pylori-positive patients showed more severe gastritis. Furthermore, the presence of cagA was related to the presence of more severe gastritis. All CagA-positive strains had Western-type CagA. In conclusion, we found that H. pylori infection is a risk factor for peptic ulcer in the Dominican Republic. Patients with cagA-positive H. pylori could be at higher risk for severe inflammation and atrophy.
Considerable progress has been made in understanding the roles of Helicobacter pylori in inflammation and gastric cancer; however, far less is known about the roles of enterohepatic Helicobacter species (EHS) in carcinogenesis and their zoonotic or pathogenic potential. We determined the prevalence of EHS infection in a cohort of geriatric rhesus monkeys in which intestinal adenocarcinoma (IAC) is common and investigated the association between EHS infection and IAC. The cohort consisted of 36 animals, 14 of which (age 26–35 years) had IAC. Of the 36 rhesus, 35 (97 %) were positive for EHS using PCR or bacterial isolation from faeces, colonic or tumour tissues. Only a single rhesus, which had IAC, was negative for EHS by all detection methods. The EHS identified by 16S rRNA sequencing in this study were from three Helicobacter taxa: Helicobacter macacae (previously rhesus monkey taxon 1), Helicobacter sp. rhesus monkey taxon 2, previously described from strain MIT 99-5507, and Helicobacter sp. rhesus monkey taxon 4, related to Helicobacter fennelliae. Thirteen of 14 monkeys with IAC were positive for either H. macacae (7/13, 54 %), EHS rhesus monkey taxon 4 (4/13, 31 %) or a mixture of the two EHS (2/13, 15 %). These results indicate that EHS are prevalent among aged rhesus macaques with IAC. Using Helicobacter genus-specific florescent in situ hybridization, EHS were detected on the surface of colonic epithelia of infected monkeys. All Helicobacter isolates, including H. macacae, effectively adhered to, invaded, and significantly induced proinflammatory genes, including IL-8, IL-6, TNF-α and iNOS, while downregulating genes involved in the function of inflammasomes, particularly IL-1β, CASPASE-1, NRLP3, NLRP6 and NLRC4 in the human colonic T84 cell line (P<0.0001). These results suggest that EHS may represent an aetiological agent mediating diarrhoea, chronic inflammation, and possibly intestinal cancer in non-human primates, and may play a role in similar disease syndromes in humans. Downregulation of inflammasome function may represent an EHS strategy for long-term persistence in the host and play a role in inducing pathological changes in the host’s lower bowel.
Proteus mirabilis is a Gram-negative enteric bacterium that causes complicated urinary tract infections, particularly in patients with indwelling catheters. Sequencing of clinical isolate P. mirabilis HI4320 revealed the presence of 17 predicted chaperone-usher fimbrial operons. We classified these fimbriae into three groups by their genetic relationship to other chaperone-usher fimbriae. Sixteen of these fimbriae are encoded by all seven currently sequenced P. mirabilis genomes. The predicted protein sequence of the major structural subunit for 14 of these fimbriae was highly conserved (≥95 % identity), whereas three other structural subunits (Fim3A, UcaA and Fim6A) were variable. Further examination of 58 clinical isolates showed that 14 of the 17 predicted major structural subunit genes of the fimbriae were present in most strains (>85 %). Transcription of the predicted major structural subunit genes for all 17 fimbriae was measured under different culture conditions designed to mimic conditions in the urinary tract. The majority of the fimbrial genes were induced during stationary phase, static culture or colony growth when compared to exponential-phase aerated culture. Major structural subunit proteins for six of these fimbriae were detected using MS of proteins sheared from the surface of broth-cultured P. mirabilis, demonstrating that this organism may produce multiple fimbriae within a single culture. The high degree of conservation of P. mirabilis fimbriae stands in contrast to uropathogenic Escherichia coli and Salmonella enterica, which exhibit greater variability in their fimbrial repertoires. These findings suggest there may be evolutionary pressure for P. mirabilis to maintain a large fimbrial arsenal.
Antimicrobial peptides are an important component of the innate immune defence. Mycobacterium avium subsp. hominissuis (M. avium) is an organism that establishes contact with the respiratory and gastrointestinal mucosa as a necessary step for infection. M. avium is resistant to high concentrations of polymyxin B, a surrogate for antimicrobial peptides. To determine gene-encoding proteins that are associated with this resistance, we screened a transposon library of M. avium strain 104 for susceptibility to polymyxin B. Ten susceptible mutants were identified and the inactivated genes sequenced. The great majority of the genes were related to cell wall synthesis and permeability. The mutants were then examined for their ability to enter macrophages and to survive macrophage killing. Three clones among the mutants had impaired uptake by macrophages compared with the WT strain, and all ten clones were attenuated in macrophages. The mutants were also shown to be susceptible to cathelicidin (LL-37), in contrast to the WT bacterium. All but one of the mutants were significantly attenuated in mice. In conclusion, this study indicated that the M. avium envelope is the primary defence against host antimicrobial peptides.
Lactobacillus species play an integral part in the health of the vaginal microbiota. We compared vaginal Lactobacillus species in women from India and the USA with and without bacterial vaginosis (BV). Between July 2009 and November 2010, a cross-sectional study was conducted among 40 women attending a women’s health clinic in Mysore, India, and a sexually transmitted diseases clinic in San Francisco, USA. Women were diagnosed with BV using Amsel’s criteria and the Nugent score. Lactobacillus 16S rDNA was sequenced to speciate the cultured isolates. Ten Indian and 10 US women without BV were compared with an equal number of women with BV. Lactobacilli were isolated from all healthy women, but from only 10 % of Indian and 50 % of US women with BV. 16S rDNA from 164 Lactobacillus colonies was sequenced from healthy women (126 colonies) and women with BV (38 colonies). Seven cultivable Lactobacillus species were isolated from 11 Indian women and nine species from 15 US women. The majority of Lactobacillus species among Indian women were L. crispatus (25 .0%), L. jensenii (25.0 %) and L. reuteri (16.7 %). Among US women, L. crispatus (32.0 %), L. jensenii (20.0 %) and L. coleohominis (12.0 %) predominated. L. jensenii and L. crispatus dominated the vaginal flora of healthy Indian and US women. Indian women appeared to have a higher percentage of obligate heterofermentative species, suggesting the need for a larger degree of metabolic flexibility and a more challenging vaginal environment.
Low levels of the vitamin D-regulated antimicrobial peptide cathelicidin (LL-37) may negatively impact the immune status of human immunodeficiency virus-1 (HIV-1) infected individuals (HIV+). We compared plasma LL-37 levels in healthy controls (HIV−) and HIV+ individuals on or off antiretroviral therapies (ARTs) (ART+ and ART−, respectively), and evaluated the relationship between vitamin D and LL-37 levels. In this cross-sectional study, levels of LL-37, 25-hydroxycholecalciferol [25(OH)D3] and 1,25-dihydroxycholecalciferol [1,25(OH)2D3] were measured from an initial cohort of 18 healthy controls and 10 HIV+/ART− individuals. Because this cohort lacked HIV+/ART+ subjects, LL-37 was also quantified from a second cohort of 10 HIV+/ART− and 13 HIV+/ART+ individuals. LL-37 levels were significantly lower in the HIV+/ART− group compared to the healthy controls (P = 0.01). A direct relationship was observed between LL-37 and both 25(OH)D3 and 1,25(OH)2D3. The level of 25(OH)D3 was predictive of higher LL-37 (P = 0.04) and for any given level of 25(OH)D3, HIV+/ART− subjects averaged 20 % lower LL-37 compared to the healthy controls (P = 0.045). For any given level of 1,25(OH)2D3, HIV+/ART− subjects averaged 25 % lower LL-37 compared to the healthy controls (P = 0.018), although 1,25(OH)2D3 was not predictive of higher LL-37 (P = 0.28). Finally, LL-37 levels were significantly lower in the HIV+/ART− group compared to the HIV+/ART+ group from the second cohort (P = 0.045). Untreated HIV infection may contribute to lower LL-37 levels, independent of vitamin D levels. ART treatment may potentially mitigate this decrease in LL-37 levels.
Biofilms represent microbial communities, encased in a self-produced matrix or extracellular polymeric substance. Microbial biofilms are likely responsible for a large proportion of clinically significant infections and the multicellular nature of biofilm existence has been repeatedly associated with antibiotic resistance. Classical in vitro antibiotic-susceptibility testing utilizes artificial growth media and planktonic microbes, but this method may not account for the variability inherent in environments subject to biofilm growth in vivo. Experiments were designed to test the hypothesis that nutrient concentration can modulate the antibiotic susceptibility of Staphylococcus aureus biofilms. Developing S. aureus biofilms initiated on surgical sutures, and in selected experiments planktonic cultures, were incubated for 16 h in 66 % tryptic soy broth, 0.2 % glucose (1× TSBg), supplemented with bactericidal concentrations of gentamicin, streptomycin, ampicillin or vancomycin. In parallel experiments, antibiotics were added to growth medium diluted one-third (1/3× TSBg) or concentrated threefold (3× TSBg). Following incubation, viable bacteria were enumerated from planktonic cultures or suture sonicates, and biofilm biomass was assayed using spectrophotometry. Interestingly, bactericidal concentrations of gentamicin (5 µg gentamicin ml−1) and streptomycin (32 µg streptomycin ml−1) inhibited biofilm formation in samples incubated in 1/3× or 1× TSBg, but not in samples incubated in 3× TSBg. The nutrient dependence of aminoglycoside susceptibility is not only associated with biofilm formation, as planktonic cultures incubated in 3× TSBg in the presence of gentamicin also showed antibiotic resistance. These findings appeared specific for aminoglycosides because biofilm formation was inhibited in all three growth media supplemented with bactericidal concentrations of the cell wall-active antibiotics, ampicillin and vancomycin. Additional experiments showed that the ability of 3× TSBg to overcome the antibacterial effects of gentamicin was associated with decreased uptake of gentamicin by S. aureus. Uptake is known to be decreased at low pH, and the kinetic change in pH of growth medium from biofilms incubated in 5 µg gentamicin ml−1 in the presence of 3× TSBg was decreased when compared with pH determinations from biofilms formed in 1/3× or 1× TSBg. These studies underscore the importance of environmental factors, including nutrient concentration and pH, on the antibiotic susceptibility of S. aureus planktonic and biofilm bacteria.
Brucellosis is a globally distributed zoonotic disease affecting animals and humans, and current antibiotic and vaccine strategies are not optimal. The surface-exposed protein Omp25 is involved in Brucella virulence and plays an important role in Brucella pathogenesis during infection, suggesting that Omp25 could be a useful target for selecting potential therapeutic molecules to inhibit Brucella pathogenesis. In this study, we identified, we believe for the first time, peptides that bind specifically to the Omp25 protein of pathogens, using a phage panning technique, After four rounds of panning, 42 plaques of eluted phages were subjected to pyrosequencing. Four phage clones that bound better than the other clones were selected following confirmation by ELISA and affinity constant determination. The peptides selected could significantly inhibit Brucella abortus 2308 (S2308) internalization and intracellular growth in RAW264.7 macrophages, and significantly induce secretion of TNF-α and IL-12 in peptide- and S2308-treated cells. Any observed peptide (OP11, OP27, OP35 or OP40) could significantly inhibit S2308 infection in BALB/c mice. Moreover, the peptide OP11 was the best candidate peptide for inhibiting S2308 infection in vitro and in vivo. These results suggest that peptide OP11 has potential for exploitation as a peptide drug in resisting S2308 infection.
Enterotoxigenic Escherichia coli (ETEC) is one of the most
common causes of diarrhoea among young children in developing countries. ETEC
vaccines offer promise in reducing the burden of ETEC disease, but the
development of these vaccines relies on the characterization of ETEC isolates
from a variety of settings. To best reflect the full spectrum of ETEC disease in
León, Nicaragua, the aim of this study was to characterize ETEC strains
isolated from children with diarrhoea attending different settings (hospital,
primary care clinics and in the community) and children from different age
groups. We characterized ETEC isolates in terms of their colonization factors
(CFs) and enterotoxins, and determined whether these factors varied with setting
and age group. Diarrhoeal stool samples were obtained from children under the
age of 60 months from: (1) the regional public hospital, (2) four public primary
care clinics, and (3) a population-based cohort. In total, 58 ETEC-positive
isolates were analysed by multiplex-PCR assays for the identification of CFs
(CS1, CS2, CS3, CS4, CS5, CS6, CS7, CS8, CS12, CS13, CS14, CS15, CS17, CS18,
CS19, CS20, CS21, CS22 and CFA/I), and enterotoxins [heat-labile toxin (LT) and
heat-stable variants STh and STp]. The frequency of CFs and enterotoxins was
compared among the three settings and for different age groups, using
Fisher’s exact test or a χ2 test. At least one CF was
detected among one-half of samples; CS19 was detected among all strains in which
a CF was identified, either alone or in combination with another CF. Among all
CFs detected, 91.7 % were identified as members of the class 5 fimbrial
family. CFs were detected more commonly among samples from infants captured in
the health facility setting compared with the community setting. Overall, LT was
detected among 67.2 % of samples, STh was detected among 20.7 %
and both enterotoxins were detected among 12.1 %. The enterotoxin STh
was detected more commonly among cases in the community, whilst a combination of
STh and LT was detected more commonly among cases treated in health facilities.
Our results suggest that, to protect against diarrhoeal cases associated with
this E. coli pathotype in León, Nicaragua, an ETEC
vaccine that effectively targets the archeotype CFA/I of the class 5 fimbrial
family would be the most effective in this setting.
Pseudomonas aeruginosa is one of the most dreaded opportunistic pathogens accounting for 10 % of hospital-acquired infections, with a 50 % mortality rate in chronically ill patients. The increased prevalence of drug-resistant isolates is a major cause of concern. Resistance in P. aeruginosa is mediated by various mechanisms, some of which are shared among different classes of antibiotics and which raise the possibility of cross-resistance. The goal of this study was to explore the effect of subinhibitory concentrations (SICs) of clinically relevant antibiotics and the role of a global antibiotic resistance and virulence regulator, AmpR, in developing cross-resistance. We investigated the induction of transient cross-resistance in P. aeruginosa PAO1 upon exposure to SICs of antibiotics. Pre-exposure to carbapenems, specifically imipenem, even at 3 ng ml−1, adversely affected the efficacy of clinically used penicillins and cephalosporins. The high β-lactam resistance was due to elevated expression of both ampC and ampR, encoding a chromosomal β-lactamase and its regulator, respectively. Differences in the susceptibility of ampR and ampC mutants suggested non-AmpC-mediated regulation of β-lactam resistance by AmpR. The increased susceptibility of P. aeruginosa in the absence of ampR to various antibiotics upon SIC exposure suggests that AmpR plays a major role in the cross-resistance. AmpR was shown previously to be involved in resistance to quinolones by regulating MexEF–OprN efflux pump. The data here further indicate the role of AmpR in cross-resistance between quinolones and aminoglycosides. This was confirmed using quantitative PCR, where expression of the mexEF efflux pump was further induced by ciprofloxacin and tobramycin, its substrate and a non-substrate, respectively, in the absence of ampR. The data presented here highlight the intricate cross-regulation of antibiotic resistance pathways at SICs of antibiotics and the need for careful assessment of the order of antibiotic regimens as this may have dire consequences. Targeting a global regulator such as AmpR that connects diverse pathways is a feasible therapeutic approach to combat P. aeruginosa pathogenesis.
A 40-year-old man was admitted to hospital with a 5 day history of fever, restlessness and altered sensorium. Peripheral blood smears showed a Plasmodium vivax and Plasmodium falciparum mixed infection as revealed by the presence of rings, schizonts and gametocyte forms of the parasites. The patient soon became unconscious due to subdural haematoma (SDH) associated with disseminated intravascular coagulation and thrombocytopenia. Immediate intervention with a right fronto-parieto temporal craniectomy, evacuation of the SDH and intravenous quinine administration resulted in the patient’s complete recovery within 8 days of admission, and he was discharged in good clinical condition.
Enteropathogenic Escherichia coli (EPEC) are a major cause of infant diarrhoea in developing countries and a significant public health issue in industrialized countries. Currently there are no simple tests available for the diagnosis of EPEC. Serology of O-antigens is widely used routinely in many laboratories throughout the world, even though it has been known for many years to be an unreliable indicator of EPEC virulence. We have developed a simple, low-cost immunodiagnostic test based on the EspA filament, an essential virulence factor of EPEC and the related enterohaemorrhagic E. coli (EHEC). Using recombinant proteins of the five major variants of EspA as immunogens, we raised a panel of three monoclonal antibodies in mice that detects all variants of the native target in bacterial cultures. The antibodies proved suitable for application in sandwich-type assays, including ELISA and lateral flow immunoassays (LFI). Prototypes for both assays were specific for EPEC and EHEC strains when tested against a panel of control micro-organisms. We have also developed a simple, affordable culture medium, A/E medium, which optimizes expression of EspA allowing improved sensitivity of detection compared with standard Dulbecco’s modified Eagle’s medium. Together these reagents form the basis of robust, informative tests for EPEC for use especially in developing countries but also for routine screening in any clinical laboratory.
We determined the in vitro antifungal activity of liposomal amphotericin B (L-AmB) against 604 clinical yeast isolates. Amphotericin B deoxycholate (D-AmB) was tested in parallel against all the isolates. Susceptibility testing was performed according to the Clinical and Laboratory Standards Institute (CLSI) M27-A3 method. Overall, L-AmB was highly active against the isolates (mean MIC, 0.42 µg ml−1; MIC90, 1 µg ml−1; 97.2 % of MICs were ≤1 µg ml−1) and comparable to D-AmB (mean MIC, 0.48 µg ml−1; MIC90, 1 µg ml−1; 97.3 % of MICs were ≤1 µg ml−1). The in vitro activity of D-AmB and L-AmB was correlated (R2 = 0.61; exp(b), 2.3; 95 % CI, 2.19–2.44, P<0.001). Candida albicans (mean MICs of D-AmB and L-AmB, 0.39 µg ml−1 and 0.31 µg ml−1, respectively) and Candida parapsilosis (mean MICs of D-AmB and L-AmB, 0.38 µg ml−1 and 0.35 µg ml−1, respectively) were the species most susceptible to the agents tested, while Candida krusei (currently named Issatchenkia orientalis) (mean MICs of D-AmB and L-AmB, 1.27 µg ml−1 and 1.13 µg ml−1, respectively) was the least susceptible. The excellent in vitro activity of L-AmB may have important implications for empirical treatment approaches and support its role in treatment of a wide range of invasive infections due to yeasts.
Acinetobacter baumannii is a Gram-negative bacterium that opportunistically infects critically ill hospitalized patients with breaches in skin integrity and airway protection, leading to significant morbidity and mortality. Considering the paucity of well-established animal models of immunosuppression to study A. baumannii pathogenesis, we set out to characterize a murine model of immunosuppression using the alkylating agent cyclophosphamide (CYP). We hypothesized that CYP-induced immunosuppression would increase the susceptibility of C57BL/6 mice to developing A. baumannii-mediated pneumonia followed by systemic disease. We demonstrated that CYP intensified A. baumannii-mediated pulmonary disease, abrogated normal immune cell function and led to altered pro-inflammatory cytokine release. The development of an animal model that mimics A. baumannii infection onset in immunosuppressed individuals is crucial for generating novel approaches to patient care and improving public health strategies to decrease exposure to infection for individuals at risk.
A series of clinical isolates of drug-resistant (DR) Acinetobacter baumannii with diverse drug susceptibility was detected from eight patients in the emergency intensive care unit of Tokai University Hospital. The initial isolate was obtained in March 2010 (A. baumannii Tokai strain 1); subsequently, seven isolates were obtained from patients (A. baumannii Tokai strains 2–8) and one isolate was obtained from an air-fluidized bed used by five of the patients during the 3 months from August to November 2011. The isolates were classified into three types of antimicrobial drug resistance patterns (RRR, SRR and SSR) according to their susceptibility (S) or resistance (R) to imipenem, amikacin and ciprofloxacin, respectively. Genotyping of these isolates by multilocus sequence typing revealed one sequence type, ST208, whilst that by a DiversiLab analysis revealed two subtypes. All the isolates were positive for blaOXA-51-like and blaOXA-66, as assessed by PCR and DNA sequencing. A. baumannii Tokai strains 1–8 and 10 (RRR, SRR and SSR) had quinolone resistance-associated mutations in gyrA/parC, as revealed by DNA sequencing. The ISAba1 upstream of blaOXA-51-like and aminoglycoside resistance-associated gene, armA, were detected in A. baumannii Tokai strains 1–7 and 10 (RRR and SRR) as assessed by PCR. Among the genes encoding resistance–nodulation–division family pumps (adeB, adeG and adeJ) and outer-membrane porins (oprD and carO), overexpression of adeB and adeJ and suppression of oprD and carO were seen in isolates of A. baumannii Tokai strain 2 (RRR), as assessed by real-time PCR. Thus, the molecular characterization of a series of isolates of DR A. baumannii revealed the outbreak of ST208 and diverse antimicrobial drug susceptibilities, which almost correlated with differential gene alterations responsible for each type of drug resistance.
Gastric cancer is the second leading cause of cancer-related mortality in the world. Recently, serum Helicobacter pylori antibodies and pepsinogen (PG) have been used for gastric cancer screening. The incidence of gastric cancer in Bhutan is reported to be quite high compared with that in neighbouring countries. In this study, 381 subjects from three areas of Bhutan were assessed for gastric mucosal atrophy and serological parameters. Anti-H. pylori IgG, PG I, PG II and cytotoxin-associated gene A (CagA) antibodies were measured using ELISA. Subjects were classified into four groups according to H. pylori and PG seropositivity: Group A (H. pylori-negative/PG-negative), Group B (H. pylori-positive/PG-negative), Group C (H. pylori-positive/PG-positive) and Group D (H. pylori-negative/PG-positive). The prevalence of H. pylori in the 381 subjects was 71.1 % (271/381), with high infection rates found in rural areas. The PG I/II ratio was significantly inversely correlated with the atrophy score in the antrum and the corpus (P<0.001). Multivariate analysis showed that the PG status was significantly associated with the presence of atrophy in the corpus. The prevalence of the PG-positive status was significantly higher among H. pylori-positive subjects than among H. pylori-negative subjects (P<0.001). Based on the ABC method, Group B was the most dominant, followed by Group A, Group C and Group D. The high incidence of gastric cancer in Bhutan can be attributed to the high prevalence of H. pylori infection and gastric mucosal atrophy.
Ventilator-associated pneumonia (VAP) is a serious healthcare-associated infection that affects up to 30 % of intubated and mechanically ventilated patients in intensive care units (ICUs) worldwide. The bacterial aetiology and corresponding antimicrobial susceptibility of VAP is highly variable, and can differ between countries, national provinces and even between different wards in the same hospital. We aimed to understand and document changes in the causative agents of VAP and their antimicrobial susceptibility profiles retrospectively over an 11 year period in a major infectious disease hospital in southern Vietnam. Our analysis outlined a significant shift from Pseudomonas aeruginosa to Acinetobacter spp. as the most prevalent bacteria isolated from quantitative tracheal aspirates in patients with VAP in this setting. Antimicrobial resistance was common across all bacterial species and we found a marked proportional annual increase in carbapenem-resistant Acinetobacter spp. over a 3 year period from 2008 (annual trend; odds ratio 1.656, P = 0.010). We further investigated the possible emergence of a carbapenem-resistant Acinetobacter baumannii clone by multiple-locus variable number tandem repeat analysis, finding a blaOXA-23-positive strain that was associated with an upsurge in the isolation of this pathogen. We additionally identified a single blaNDM-1-positive A. baumannii isolate. This work highlights the emergence of a carbapenem-resistant clone of A. baumannii and a worrying trend of antimicrobial resistance in the ICU of the Hospital for Tropical Diseases in Ho Chi Minh City, Vietnam.
Autotransporter protein secretion represents one of the simplest forms of secretion across Gram-negative bacterial membranes. Once secreted, autotransporter proteins either remain tethered to the bacterial surface or are released following proteolytic cleavage. Autotransporters possess a diverse array of virulence-associated functions such as motility, cytotoxicity, adherence and autoaggregation. To better understand the role of autotransporters in disease, our research focused on the autotransporters of Yersinia pestis, the aetiological agent of plague. Y. pestis strain CO92 has nine functional conventional autotransporters, referred to as Yaps for Yersinia
autotransporter proteins. Three Yaps have been directly implicated in virulence using established mouse models of plague infection (YapE, YapJ and YapK). Whilst previous studies from our laboratory have shown that most of the CO92 Yaps are cell associated, YapE and YapG are processed and released by the omptin protease Pla. In this study, we identified the Pla cleavage sites in YapG that result in many released forms of YapG in Y. pestis, but not in the evolutionarily related gastrointestinal pathogen, Yersinia pseudotuberculosis, which lacks Pla. Furthermore, we showed that YapG does not contribute to Y. pestis virulence in established mouse models of bubonic and pneumonic infection. As Y. pestis has a complex life cycle involving a wide range of mammalian hosts and a flea vector for transmission, it remains to be elucidated whether YapG has a measurable role in any other stage of plague disease.
Rickettsia conorii, the causative agent of Mediterranean spotted fever, preferentially infects human microvascular endothelium and activates pro-inflammatory innate immune responses as evidenced by enhanced expression and secretion of cytokines and chemokines. Our recent studies reveal that human microvascular endothelial cells (HMECs) infected with R. conorii also launch ‘antiviral’ host defence mechanisms typically governed by type I interferons. To summarize, infected HMECs secrete IFN-β to activate STAT1 in an autocrine/paracrine manner and display increased expression of IFN-stimulated genes, for example ISG15, which in turn activate innate responses to interfere with intracellular replication of rickettsiae. We now present evidence that UBP43 and SOCS1, known negative regulators of JAK/STAT signalling, are also induced in R. conorii-infected HMECs, of which UBP43 but not SOCS1 functions to negatively regulate STAT1 activation. Interestingly, UBP43 induction is almost completely abolished in the presence of IFN-β-neutralizing antibody, implicating an important role for UBP43 as a feedback inhibitor for IFN-β-mediated STAT1 activation. In contrast, SOCS1 expression is only partially affected by IFN-β neutralization, implicating potential involvement of as-yet-unidentified IFN-independent mechanism(s) in SOCS1 induction during R. conorii infection. A number of IFN-stimulated genes, including ISG15, OAS1, MX1, IRF1, IRF9 and TAP1 are also induced in an IFN-β-dependent manner, whereas GBP1 remains unaffected by IFN-β neutralization. Increased STAT1 phosphorylation in HMECs subjected to UBP43 knockdown led to transcriptional activation of OAS1, MX1 and GBP1, confirming the negative regulatory role of UBP43. Although IRF1, IRF9 and TAP1 were induced by IFN-β, siRNA-mediated silencing of UBP43 or SOCS1 did not significantly affect their transcriptional activation. Expression of ISG15 was, however, increased in HMECs transfected with siRNA for UBP43 and SOCS1. Thus, unique regulatory patterns of induced expression of UBP43, SOCS1 and IFN-stimulated genes represent pathogen-specific responses underlying IFN-β-mediated host endothelial signalling during the pathogenesis of spotted fever group rickettsiosis.
Cholera toxin (CT), the principal virulence factor secreted by Vibrio cholerae, is an A-B5 type exotoxin that binds to host cell GM1-gangliosides and is responsible for cholera diarrhoea. We tested the hypothesis that the cyclic hexasaccharide α-cyclodextrin (α-CD), but not the cyclic heptasaccharides methyl-β-cyclodextrin (MD-β-CD) and hydroxypropyl-β-cyclodextrin (HP-β-CD) inhibit binding of CT to GM1-gangliosides. We report that α-CD decreases CT binding to GM1-ganglioside-coated microtitre plate wells and on the surface of fixed HeLa cells in a concentration-dependent manner, suggesting that this may be a promising lead for the development of compounds with therapeutic properties.
Enteroaggregative Escherichia coli (EAEC) is increasingly recognized as a common cause of diarrhoea in healthy, malnourished and immune-deficient adults and children. There is no reproducible non-neonatal animal model for longitudinal studies of disease mechanism or therapy. Using two strains of human-derived EAEC to challenge weaned C57BL/6 mice, we explored an in vivo model of EAEC infection in mice, in which disease was monitored quantitatively as the growth rate, stool shedding and tissue burden of organisms; nutritional status was varied, and a new class of therapeutics was assessed. A single oral challenge of EAEC strain 042 resulted in significant growth shortfalls (5–8 % of body weight in 12 days), persistent shedding of micro-organisms in stools [>103.2 c.f.u. (10 mg stool)−1 for at least 14 days] and intestinal tissue burden [~103 c.f.u. (10 mg tissue)−1 detectable up to 14 days post-challenge]. Moderate malnourishment of mice using a ‘regional basic diet’ containing 7 % protein and reduced fat and micronutrients heightened all parameters of infection. Nitazoxanide in subMIC doses, administered for 3 days at the time of EAEC challenge, lessened growth shortfalls (by >10 % of body weight), stool shedding [by 2–3 logs (10 mg stool)−1] and tissue burden of organisms (by >75 % in the jejunum and colon). Thus, weaned C57BL/6 mice challenged with EAEC is a convenient, readily inducible model of EAEC infection with three highly quantifiable outcomes in which disease severity is dependent on the nutritional status of the host, and which is modifiable in the presence of inhibitors of pyruvate ferredoxin oxidoreductase such as nitazoxanide.
Lipo-oligosaccharide (LOS) is a major surface component and virulence factor of the human respiratory pathogen Moraxella catarrhalis. Two late acyltransferase genes, lpxX and lpxL, have been identified involved in the incorporation of acyloxyacyl-linked secondary acyl chains into lipid A during M. catarrhalis LOS biosynthesis. In this study, a double mutant with a deletion of both the lpxX and lpxL genes in M. catarrhalis strain O35E was constructed and named O35ElpxXL. Structural analysis of lipid A showed that the O35ElpxXL mutant lacked two decanoic acids (10 : 0) and one dodecanoic (lauric) acid (12 : 0). In comparison with the O35E parental strain and the single mutants O35ElpxX and O35ElpxL, the double mutant O35ElpxXL displayed prominently decreased endotoxin content, reduced resistance to normal human serum and accelerated bacterial clearance at 0, 3 and 6 h after an aerosol challenge in a mouse model of bacterial pulmonary clearance. These results indicate that these two genes encoding late acyltransferases responsible for lipid A biosynthesis jointly contribute to the biological activities and pathogenicity of M. catarrhalis. The double mutant O35ElpxXL with dramatically reduced toxicity is proposed as a potential vaccine candidate against M. catarrhalis infections for further investigation.