Leptospirosis is a globally distributed bacterial infectious disease caused by pathogenic members of the genus Leptospira. Infection can lead to illness ranging from mild and non-specific to severe, with jaundice, kidney and liver dysfunction, and widespread endothelial damage. The adhesion of pathogenic Leptospira species (spp.), the causative agent of leptospirosis, to host tissue components is necessary for infection and pathogenesis. While it is well-established that extracellular matrix (ECM) components play a role in the interaction of the pathogen with host molecules, we have shown that pathogenic Leptospira interrogans binds to host cells more efficiently than to ECM components. Using in vitro phage display to select for phage clones that bind to EA.hy926 endothelial cells, we identified the putative lipoproteins LIC10508 and LIC13411, and the conserved hypothetical proteins LIC12341 and LIC11574, as candidate L. interrogans sv. Copenhageni st. Fiocruz L1-130 adhesins. Recombinant LIC11574, but not its L. biflexa homologue LBF1629, exhibited dose-dependent binding to both endothelial and epithelial cells. In addition, LIC11574 and LIC13411 bind to VE-cadherin, an endothelial cell receptor for L. interrogans. Extraction of bacteria with the non-ionic detergent Triton X-114 resulted in partitioning of the candidate adhesins to the detergent fraction, a likely indication that these proteins are outer membrane localized. All candidate adhesins were recognized by sera obtained from leptospirosis patients but not by sera from healthy individuals as assessed by western blot. This work has identified bacterial adhesins that are potentially involved in L. interrogans infection of the mammalian host, and through cadherin binding, may contribute to dissemination and vascular damage. Our findings may be of value in leptospirosis control and prevention, with the bacterial adhesins potentially serving as targets for development of diagnostics, therapeutics, and vaccines.

Background

The immune system depends on effector pathways to eliminate invading pathogens from the host in vivo. Macrophages (MΦ) of the innate immune system are armed with vitamin D-dependent antimicrobial responses to kill intracellular microbes. However, how the physiological levels of vitamin D during MΦ differentiation affect phenotype and function is unknown.

Methodology/principal

The human innate immune system consists of divergent MΦ subsets that serve distinct functions in vivo. Both IL-15 and IL-10 induce MΦ differentiation, but IL-15 induces primary human monocytes to differentiate into antimicrobial MΦ (IL-15 MΦ) that robustly express the vitamin D pathway. However, how vitamin D status alters IL-15 MΦ phenotype and function is unknown. In this study, we found that adding 25-hydroxyvitamin D3 (25D3) during the IL-15 induced differentiation of monocytes into MΦ increased the expression of the antimicrobial peptide cathelicidin, including both CAMP mRNA and the encoded protein cathelicidin in a dose-dependent manner. The presence of physiological levels of 25D during differentiation of IL-15 MΦ led to a significant vitamin D-dependent antimicrobial response against intracellular Mycobacterium leprae but did not change the phenotype or phagocytic function of these MΦ. These data suggest that activation of the vitamin D pathway during IL-15 MΦ differentiation augments the antimicrobial response against M. leprae infection.

Conclusions/significance

Our data demonstrates that the presence of vitamin D during MΦ differentiation bestows the capacity to mount an antimicrobial response against M. leprae.

Background

Noma is a gangrenous disease that leads to severe disfigurement of the face with high morbidity and mortality, but its etiology remains unknown. Young children in developing countries are almost exclusively affected. The purpose of the study was to record and compare bacterial diversity in oral samples from children with or without acute noma or acute necrotizing gingivitis from a defined geographical region in Niger by culture-independent molecular methods.

Methods and principal findings

Gingival samples from 23 healthy children, nine children with acute necrotizing gingivitis, and 23 children with acute noma (both healthy and diseased oral sites) were amplified using "universal" PCR primers for the 16 S rRNA gene and pooled according to category (noma, healthy, or acute necrotizing gingivitis), gender, and site status (diseased or control site). Seven libraries were generated. A total of 1237 partial 16 S rRNA sequences representing 339 bacterial species or phylotypes at a 98-99% identity level were obtained. Analysis of bacterial composition and frequency showed that diseased (noma or acute necrotizing gingivitis) and healthy site bacterial communities are composed of similar bacteria, but differ in the prevalence of a limited group of phylotypes. Large increases in counts of Prevotella intermedia and members of the Peptostreptococcus genus are associated with disease. In contrast, no clear-cut differences were found between noma and non-noma libraries.

Conclusions

Similarities between acute necrotizing gingivitis and noma samples support the hypothesis that the disease could evolve from acute necrotizing gingivitis in certain children for reasons still to be elucidated. This study revealed oral microbiological patterns associated with noma and acute necrotizing gingivitis, but no evidence was found for a specific infection-triggering agent.

We aim to understand the microbial ecology of noma (cancrum oris), a devastating ancient illness which causes severe facial disfigurement in>140,000 malnourished children every year. The cause of noma is still elusive. A chaotic mix of microbial infection, oral hygiene and weakened immune system likely contribute to the development of oral lesions. These lesions are a plausible entry point for unidentified microorganisms that trigger gangrenous facial infections. To catalog bacteria present in noma lesions and identify candidate noma-triggering organisms, we performed a cross-sectional sequencing study of 16S rRNA gene amplicons from sixty samples of gingival fluid from twelve healthy children, twelve children suffering from noma (lesion and healthy sites), and twelve children suffering from Acute Necrotizing Gingivitis (ANG) (lesion and healthy sites). Relative to healthy individuals, samples taken from lesions in diseased mouths were enriched with Spirochaetes and depleted for Proteobacteria. Samples taken from healthy sites of diseased mouths had proportions of Spirochaetes and Proteobacteria that were similar to healthy control individuals. Samples from noma mouths did not have a higher abundance of Fusobacterium, casting doubt on its role as a causative agent of noma. Microbial communities sampled from noma and ANG lesions were dominated by the same Prevotella intermedia OTU, which was much less abundant in healthy sites sampled from the same mouths. Multivariate analysis confirmed that bacterial communities in healthy and lesion sites were significantly different. Several OTUs in the Orders Erysipelotrichales, Clostridiales, Bacteroidales, and Spirochaetales were identified as indicators of noma, suggesting that one or more microbes within these Orders is associated with the development of noma lesions. Future studies should include longitudinal sampling of viral and microbial components of this community, before and early in noma lesion development.

Background

Brucellosis, a zoonotic infection caused by one of the Gram-negative intracellular bacteria of the Brucella genus, is an ongoing public health problem in Perú. While most patients who receive standard antibiotic treatment recover, 5-40% suffer a brucellosis relapse. In this study, we examined the ex vivo immune cytokine profiles of recovered patients with a history of acute and relapsing brucellosis.

Methodology/principal findings

Blood was taken from healthy control donors, patients with a history of acute brucellosis, or patients with a history of relapsing brucellosis. Peripheral blood mononuclear cells were isolated and remained in culture without stimulation or were stimulated with a panel of toll-like receptor agonists or heat-killed Brucella melitensis (HKBM) isolates. Innate immune cytokine gene expression and protein secretion were measured by quantitative real-time polymerase chain reaction and a multiplex bead-based immunoassay, respectively. Acute and relapse patients demonstrated consistently elevated cytokine gene expression and secretion levels compared to controls. Notably, these include: basal and stimulus-induced expression of GM-CSF, TNF-α, and IFN-γ in response to LPS and HKBM; basal secretion of IL-6, IL-8, and TNF-α; and HKBM or Rev1-induced secretion of IL-1β, IL-2, GM-CSF, IFN-Υ, and TNF-α. Although acute and relapse patients were largely indistinguishable by their cytokine gene expression profiles, we identified a robust cytokine secretion signature that accurately discriminates acute from relapse patients. This signature consists of basal IL-6 secretion, IL-1β, IL-2, and TNF-α secretion in response to LPS and HKBM, and IFN-γ secretion in response to HKBM.

Conclusions/significance

This work demonstrates that informative cytokine variations in brucellosis patients can be detected using an ex vivo assay system and used to identify patients with differing infection histories. Targeted diagnosis of this signature may allow for better follow-up care of brucellosis patients through improved identification of patients at risk for relapse.

Following infection, virulent mycobacteria persist and grow within the macrophage, suggesting that the intrinsic activation of an innate antimicrobial response is subverted by the intracellular pathogen. For Mycobacterium leprae, the intracellular bacterium that causes leprosy, the addition of exogenous innate or adaptive immune ligands to the infected monocytes/macrophages was required to detect a vitamin D-dependent antimicrobial activity. We investigated whether there is an intrinsic immune response to M. leprae in macrophages that is inhibited by the pathogen. Upon infection of monocytes with M. leprae, there was no upregulation of CYP27B1 nor its enzymatic activity converting the inactive prohormone form of vitamin D (25-hydroxyvitamin D) to the bioactive form (1,25α-dihydroxyvitamin D). Given that M. leprae-induced type I interferon (IFN) inhibited monocyte activation, we blocked the type I IFN receptor (IFNAR), revealing the intrinsic capacity of monocytes to recognize M. leprae and upregulate CYP27B1. Consistent with these in vitro studies, an inverse relationship between expression of CYP27B1 vs. type I IFN downstream gene OAS1 was detected in leprosy patient lesions, leading us to study cytokine-derived macrophages (MΦ) to model cellular responses at the site of disease. Infection of IL-15-derived MΦ, similar to MΦ in lesions from the self-limited form of leprosy, with M. leprae did not inhibit induction of the vitamin D antimicrobial pathway. In contrast, infection of IL-10-derived MΦ, similar to MΦ in lesions from patients with the progressive form of leprosy, resulted in induction of type I IFN and suppression of the vitamin D directed pathway. Importantly, blockade of the type I IFN response in infected IL-10 MΦ decreased M. leprae viability. These results indicate that M. leprae evades the intrinsic capacity of human monocytes/MΦ to activate the vitamin D-mediated antimicrobial pathway via the induction of type I IFN.

Background

Leptospirosis, a re-emerging disease of global importance caused by pathogenic Leptospira spp., is considered the world's most widespread zoonotic disease. Rats serve as asymptomatic carriers of pathogenic Leptospira and are critical for disease spread. In such reservoir hosts, leptospires colonize the kidney, are shed in the urine, persist in fresh water and gain access to a new mammalian host through breaches in the skin.

Methodology/principal findings

Previous studies have provided evidence for post-translational modification (PTM) of leptospiral proteins. In the current study, we used proteomic analyses to determine the presence of PTMs on the highly abundant leptospiral protein, LipL32, from rat urine-isolated L. interrogans serovar Copenhageni compared to in vitro-grown organisms. We observed either acetylation or tri-methylation of lysine residues within multiple LipL32 peptides, including peptides corresponding to regions of LipL32 previously identified as epitopes. Intriguingly, the PTMs were unique to the LipL32 peptides originating from in vivo relative to in vitro grown leptospires. The identity of each modified lysine residue was confirmed by fragmentation pattern analysis of the peptide mass spectra. A synthetic peptide containing an identified tri-methylated lysine, which corresponds to a previously identified LipL32 epitope, demonstrated significantly reduced immunoreactivity with serum collected from leptospirosis patients compared to the peptide version lacking the tri-methylation. Further, a subset of the identified PTMs are in close proximity to the established calcium-binding and putative collagen-binding sites that have been identified within LipL32.

Conclusions/significance

The exclusive detection of PTMs on lysine residues within LipL32 from in vivo-isolated L. interrogans implies that infection-generated modification of leptospiral proteins may have a biologically relevant function during the course of infection. Although definitive determination of the role of these PTMs must await further investigations, the reduced immune recognition of a modified LipL32 epitope suggests the intriguing possibility that LipL32 modification represents a novel mechanism of immune evasion within Leptospira.

Human brucellosis is most commonly diagnosed by serology based on agglutination of fixed Brucella abortus as antigen. Nucleic acid amplification techniques have not proven capable of reproducibly and sensitively demonstrating the presence of Brucella DNA in clinical specimens. We sought to optimize a monoclonal antibody-based assay to detect Brucella melitensis lipopolysaccharide in blood by conjugating B. melitensis LPS to keyhole limpet hemocyanin, an immunogenic protein carrier to maximize IgG affinity of monoclonal antibodies. A panel of specific of monoclonal antibodies was obtained that recognized both B. melitensis and B. abortus lipopolysaccharide epitopes. An antigen capture assay was developed that detected B. melitensis in the blood of experimentally infected mice and, in a pilot study, in naturally infected Peruvian subjects. As a proof of principle, a majority (7/10) of the patients with positive blood cultures had B. melitensis lipopolysaccharide detected in the initial blood specimen obtained. One of 10 patients with relapsed brucellosis and negative blood culture had a positive serum antigen test. No seronegative/blood culture negative patients had a positive serum antigen test. Analysis of the pair of monoclonal antibodies (2D1, 2E8) used in the capture ELISA for potential cross-reactivity in the detection of lipopolysaccharides of E. coli O157:H7 and Yersinia enterocolitica O9 showed specificity for Brucella lipopolysaccharide. This new approach to develop antigen-detection monoclonal antibodies against a T cell-independent polysaccharide antigen based on immunogenic protein conjugation may lead to the production of improved rapid point-of-care-deployable assays for the diagnosis of brucellosis and other infectious diseases.

Background

Infectious Leptospira colonize the kidneys of reservoir (e.g. rats) and accidental hosts such as humans. The renal response to persistent leptospiral colonization, as measured by urinary protein biosignatures, has not been systematically studied. Urinary exosomes--bioactive membrane-bound nanovesicles--contain cell-state specific cargo that additively reflect formation all along the nephron. We hypothesized that Leptospira-infection will alter the content of urine exosomes, and further, that these Leptospira-induced alterations will hold clues to unravel novel pathways related to bacterial-host interactions.

Methodology/principal findings

Exosome protein content from 24 hour urine samples of Leptospira-infected rats was compared with that of uninfected rats using SDS-PAGE and liquid chromatography/tandem mass spectrometry (LC-MS/MS). Statistical models were used to identify significantly dysregulated proteins in Leptospira-infected and uninfected rat urine exosomes. In all, 842 proteins were identified by LC-MS/MS proteomics of total rat urine and 204 proteins associated specifically with exosomes. Multivariate analysis showed that 25 proteins significantly discriminated between uninfected control and infected rats. Alanyl (membrane) aminopeptidase, also known as CD13 topped this list with the highest score, a finding we validated by Western immunoblotting. Whole urine analysis showed Tamm-Horsfall protein level reduction in the infected rat urine. Total urine and exosome proteins were significantly different in male vs. female infected rats.

Conclusions

We identified exosome-associated renal tubule-specific responses to Leptospira infection in a rat chronic colonization model. Quantitative differences in infected male and female rat urine exosome proteins vs. uninfected controls suggest that urine exosome analysis identifies important differences in kidney function that may be of clinical and pathological significance.