Responses of Human Endothelial Cells to Pathogenic and Non-Pathogenic Leptospira Species

Leptospirosis is a widespread zoonotic infection that primarily affects residents of tropical regions, but causes infections in animals and humans in temperate regions as well. The agents of leptospirosis comprise several members of the genus Leptospira, which also includes non-pathogenic, saprophytic species. Leptospirosis can vary in severity from a mild, non-specific illness to severe disease that includes multi-organ failure and widespread endothelial damage and hemorrhage. To begin to investigate how pathogenic leptospires affect endothelial cells, we compared the responses of two endothelial cell lines to infection by pathogenic versus non-pathogenic leptospires. Microarray analyses suggested that pathogenic L. interrogans and non-pathogenic L. biflexa triggered changes in expression of genes whose products are involved in cellular architecture and interactions with the matrix, but that the changes were in opposite directions, with infection by L. biflexa primarily predicted to increase or maintain cell layer integrity, while L. interrogans lead primarily to changes predicted to disrupt cell layer integrity. Neither bacterial strain caused necrosis or apoptosis of the cells even after prolonged incubation. The pathogenic L. interrogans, however, did result in significant disruption of endothelial cell layers as assessed by microscopy and the ability of the bacteria to cross the cell layers. This disruption of endothelial layer integrity was abrogated by addition of the endothelial protective drug lisinopril at physiologically relevant concentrations. These results suggest that, through adhesion of L. interrogans to endothelial cells, the bacteria may disrupt endothelial barrier function, promoting dissemination of the bacteria and contributing to severe disease manifestations. In addition, supplementing antibiotic therapy with lisinopril or derivatives with endothelial protective activities may decrease the severity of leptospirosis

Hematogenous dissemination of pathogenic and non-pathogenic Leptospira in a short-term murine model of infection

Leptospirosis is an emerging zoonosis caused by pathogenic Leptospira spp. Because rodents are natural hosts of Leptospira, rodent models of pathogenesis have been limited, but are valuable to understand infection in reservoir animals even in the absence of disease. Mouse models of infection provide advantages due to genetic tractability, so developing murine models of Leptospira infection is crucial for further understanding the biology of this organism. Previously our laboratory developed a short-term murine model of Borrelia burgdorferi hematogenous dissemination to investigate the role of adhesion proteins on bacterial survival and dissemination within a host. Here we adapt this model to Leptospira. C3H/HeJ mice are anesthetized, inoculated intravenously, and then bacteria are allowed to circulate for up to twenty-four hours. Mice are euthanized, perfused with saline, and tissues are harvested for culture and DNA purification. Bacterial burdens are determined by quantitative PCR. Reproducible burdens of bacteria were found in tissues upon inoculation with pathogens and non-pathogens, demonstrating the utility of this model to probe different Leptospira species and strains. Pathogenic L. interrogans has a significantly higher burden in blood, liver, kidney, and bladder at one-hour post-inoculation when compared to non-pathogenic L. biflexa. Colonization of the kidney is essential to the life cycle of pathogenic Leptospira in nature. Measurable burdens of non-pathogenic L. biflexa were found in numerous organs and live leptospires were recovered from blood samples for at least three hours post-inoculation, contrary to the previous belief that non-pathogenic leptospires are rapidly cleared. This short-term murine model of Leptospira hematogenous dissemination will allow for the interrogation of virulence factors potentially important for tissue colonization and evasion of host defenses, and represents a novel animal model for investigating determinants of Leptospira infection

Molecular Mechanisms Involved in Vascular Interactions of the Lyme Disease Pathogen in a Living Host

Hematogenous dissemination is important for infection by many bacterial pathogens, but is poorly understood because of the inability to directly observe this process in living hosts at the single cell level. All disseminating pathogens must tether to the host endothelium despite significant shear forces caused by blood flow. However, the molecules that mediate tethering interactions have not been identified for any bacterial pathogen except E. coli, which tethers to host cells via a specialized pillus structure that is not found in many pathogens. Furthermore, the mechanisms underlying tethering have never been examined in living hosts. We recently engineered a fluorescent strain of Borrelia burgdorferi, the Lyme disease pathogen, and visualized its dissemination from the microvasculature of living mice using intravital microscopy. We found that dissemination was a multistage process that included tethering, dragging, stationary adhesion and extravasation. In the study described here, we used quantitative real-time intravital microscopy to investigate the mechanistic features of the vascular interaction stage of B. burgdorferi dissemination. We found that tethering and dragging interactions were mechanistically distinct from stationary adhesion, and constituted the rate-limiting initiation step of microvascular interactions. Surprisingly, initiation was mediated by host Fn and GAGs, and the Fn- and GAG-interacting B. burgdorferi protein BBK32. Initiation was also strongly inhibited by the low molecular weight clinical heparin dalteparin. These findings indicate that the initiation of spirochete microvascular interactions is dependent on host ligands known to interact in vitro with numerous other bacterial pathogens. This conclusion raises the intriguing possibility that fibronectin and GAG interactions might be a general feature of hematogenous dissemination by other pathogens

High Prevalence of Spirochetosis in Cholera Patients, Bangladesh

The microbes that accompany the etiologic agent of cholera, Vibrio cholerae, are only now being defined. In this study, spirochetes from the genus Brachyspira were identified at high titers in more than one third of cholera patients in Bangladesh. Spirochetosis should now be tracked in the setting of cholera outbreaks

Иммунологические предикторы отторжения почечного трансплантата в раннем послеоперационном периоде

ПОЧЕК ТРАНСПЛАНТАЦИЯПЕРЕСАДКА ПОЧКИПОЧЕЧНАЯ ТРАНСПЛАНТАЦИЯПОСЛЕОПЕРАЦИОННЫЙ ПЕРИОДТРАНСПЛАНТАЦИОННАЯ ИММУНОЛОГИЯТРАНСПЛАНТАТА ОТТОРЖЕНИЕДЕНДРИТНЫЕ КЛЕТКИДЕНДРИТИЧЕСКИЕ КЛЕТКИLIN-HLA-DR+CD11C+CD123-LIN-HLA-DR+CD11C-CD123+Цель. Выявить иммунологические предикторы отторжения почечного трансплантата в раннем послеоперационном периоде. Материал и методы. Из 197 реципиентов почечного трансплантата были сформированы 3 группы. Группа ПФТ (n=101) – пациенты с удовлетворительной первичной функцией трансплантата. Группа ДФТ (n=82) – пациенты с первичной дисфункцией трансплантата без эпизодов отторжения. Группа ОПТ (n=14) – пациенты с первичной дисфункцией и отторжением почечного трансплантата. Ранняя функция почечного трансплантата оценивалась на 7-е сутки после операции по уровню креатинина крови. При показателях ниже 300 мкмоль/л функция считалась первичной, при значениях, равных или превышающих 300 мкмоль/л, а также при возникновении необходимости в диализе на первой неделе после трансплантации состояние классифицировалось как дисфункция почечного трансплантата. В раннем послеоперационном периоде определяли количество дендритных клеток LIN-HLA-DR+ с фенотипом LIN-HLA-DR+CD11c+CD123- (mDC) и LIN-HLA-DR+CD11c-CD123+ (pDC) в жидкости из дренажа, установленного к почечному трансплантату во время операции. С целью прогнозирования отторжения почечного трансплантата были определены предиктивные характеристики уровня mDC и pDC в дренажной жидкости и выявлены диагностические возможности данного показателя. Результаты. Выявлено, что отторжение почечного трансплантата характеризуется значимым ростом общего числа дендритных клеток (ДК) в дренажной жидкости, преимущественно за счет миелоидных. Определены предиктивные характеристики по уровню миелоидных и плазмацитоидных ДК в дренажной жидкости. Точка отсечения уровня миелоидных дендритных клеток определена на уровне 60,32%, а для плазмацитоидных соответствовала 39,68%. Заключение. При уровне миелоидных дендритных клеток в дренажной жидкости более либо равном 60,32%, а плазмацитоидных менее либо равном 39,68% прогнозируется отторжение почечного трансплантата с чувствительностью 99% и 93% соответственно и специфичностью 89% и 91% соответственно.Objective. To determine the immunological predictors of renal graft rejection in the early postoperative period. Methods. Three groups were formed out of the 197 renal graft recipients. The group PGF (n=101) was made up of patients with satisfactory primary graft function. The group PGD (n = 82) included patients with primary graft dysfunction without episodes of rejection. The group RGR (n=14) consisted of patients with primary dysfunction and renal graft rejection. On the 7th day after transplantation the early kidney graft function was assessed on the basis of serum creatinine levels. When the serum creatinine value was lower than 300 mol/L the function was considered to be primary, at a creatinine concentration was equal to or higher than 300 mol/L, as well as in the case of need for maintenance dialysis on the first week after transplantation, the state was classified as the renal graft dysfunction. In the early postoperative period, the number of LIN-HLA-DR+ dendritic cells with the LIN- HLA-DR+CD11c+CD123- and LIN-HLA-DR+CD11c-CD123+ phenotypes in the fluid from the drainage installed to the kidney graft during surgery was determined. Predictive characteristics of the mDC and pDC levels in the drainage fluid were determined to predict renal graft rejection, and diagnostic capability of this indicator were identified. Results. It has been revealed that renal graft rejection is characterized by a significant growth of the total number of dendritic cells in the drainage fluid, mainly due to myeloid ones. Predictive characteristics were determined by the level of myeloid and plasmacytoid dendritic cells in the drainage fluid. The cut-off point of the level of myeloid dendritic cells was determined at the level of 60.32%, and for plasmacytoid dendritic cells it corresponded to 39.68%. Conclusion. With the level of myeloid dendritic cells in the drainage fluid greater or equal 60.32%, and plasmacytoid cells lower or equal 39.68%, renal graft rejection is predicted with a sensitivity of 99% and 93%, respectively, and a specificity of 89% and 91%, respectively

Real-Time High Resolution 3D Imaging of the Lyme Disease Spirochete Adhering to and Escaping from the Vasculature of a Living Host

Pathogenic spirochetes are bacteria that cause a number of emerging and re-emerging diseases worldwide, including syphilis, leptospirosis, relapsing fever, and Lyme borreliosis. They navigate efficiently through dense extracellular matrix and cross the blood–brain barrier by unknown mechanisms. Due to their slender morphology, spirochetes are difficult to visualize by standard light microscopy, impeding studies of their behavior in situ. We engineered a fluorescent infectious strain of Borrelia burgdorferi, the Lyme disease pathogen, which expressed green fluorescent protein (GFP). Real-time 3D and 4D quantitative analysis of fluorescent spirochete dissemination from the microvasculature of living mice at high resolution revealed that dissemination was a multi-stage process that included transient tethering-type associations, short-term dragging interactions, and stationary adhesion. Stationary adhesions and extravasating spirochetes were most commonly observed at endothelial junctions, and translational motility of spirochetes appeared to play an integral role in transendothelial migration. To our knowledge, this is the first report of high resolution 3D and 4D visualization of dissemination of a bacterial pathogen in a living mammalian host, and provides the first direct insight into spirochete dissemination in vivo

Tick Histamine Release Factor Is Critical for Ixodes scapularis Engorgement and Transmission of the Lyme Disease Agent

Ticks are distributed worldwide and affect human and animal health by transmitting diverse infectious agents. Effective vaccines against most tick-borne pathogens are not currently available. In this study, we characterized a tick histamine release factor (tHRF) from Ixodes scapularis and addressed the vaccine potential of this antigen in the context of tick engorgement and B. burgdorferi transmission. Results from western blotting and quantitative Reverse Transcription-PCR showed that tHRF is secreted in tick saliva, and upregulated in Borrelia burgdorferi-infected ticks. Further, the expression of tHRF was coincident with the rapid feeding phase of the tick, suggesting a role for tHRF in tick engorgement and concomitantly, for efficient B. burgdorferi transmission. Silencing tHRF by RNA interference (RNAi) significantly impaired tick feeding and decreased B. burgdorferi burden in mice. Interfering with tHRF by actively immunizing mice with recombinant tHRF, or passively transferring tHRF antiserum, also markedly reduced the efficiency of tick feeding and B. burgdorferi burden in mice. Recombinant tHRF was able to bind to host basophils and stimulate histamine release. Therefore, we speculate that tHRF might function in vivo to modulate vascular permeability and increase blood flow to the tick bite-site, facilitating tick engorgement. These findings suggest that blocking tHRF might offer a viable strategy to complement ongoing efforts to develop vaccines to block tick feeding and transmission of tick-borne pathogens

Positive Signature-Tagged Mutagenesis in Pseudomonas aeruginosa: Tracking Patho-Adaptive Mutations Promoting Airways Chronic Infection

The opportunistic pathogen Pseudomonas aeruginosa can establish life-long chronic infections in the airways of cystic fibrosis (CF) patients. Persistent lifestyle is established with P. aeruginosa patho-adaptive variants, which are clonal with the initially-acquired strains. Several reports indicated that P. aeruginosa adapts by loss-of-function mutations which enhance fitness in CF airways and sustain its clonal expansion during chronic infection. To validate this model of P. aeruginosa adaptation to CF airways and to identify novel genes involved in this microevolution, we designed a novel approach of positive-selection screening by PCR-based signature-tagged mutagenesis (Pos-STM) in a murine model of chronic airways infection. A systematic positive-selection scheme using sequential rounds of in vivo screenings for bacterial maintenance, as opposed to elimination, generated a list of genes whose inactivation increased the colonization and persistence in chronic airways infection. The phenotypes associated to these Pos-STM mutations reflect alterations in diverse aspects of P. aeruginosa biology which include lack of swimming and twitching motility, lack of production of the virulence factors such as pyocyanin, biofilm formation, and metabolic functions. In addition, Pos-STM mutants showed altered invasion and stimulation of immune response when tested in human respiratory epithelial cells, indicating that P. aeruginosa is prone to revise the interaction with its host during persistent lifestyle. Finally, sequence analysis of Pos-STM genes in longitudinally P. aeruginosa isolates from CF patients identified signs of patho-adaptive mutations within the genome. This novel Pos-STM approach identified bacterial functions that can have important clinical implications for the persistent lifestyle and disease progression of the airway chronic infection

A Chromosomally Encoded Virulence Factor Protects the Lyme Disease Pathogen against Host-Adaptive Immunity

Borrelia burgdorferi, the bacterial pathogen of Lyme borreliosis, differentially expresses select genes in vivo, likely contributing to microbial persistence and disease. Expression analysis of spirochete genes encoding potential membrane proteins showed that surface-located membrane protein 1 (lmp1) transcripts were expressed at high levels in the infected murine heart, especially during early stages of infection. Mice and humans with diagnosed Lyme borreliosis also developed antibodies against Lmp1. Deletion of lmp1 severely impaired the pathogen's ability to persist in diverse murine tissues including the heart, and to induce disease, which was restored upon chromosomal complementation of the mutant with the lmp1 gene. Lmp1 performs an immune-related rather than a metabolic function, as its deletion did not affect microbial persistence in immunodeficient mice, but significantly decreased spirochete resistance to the borreliacidal effects of anti-B. burgdorferi sera in a complement-independent manner. These data demonstrate the existence of a virulence factor that helps the pathogen evade host-acquired immune defense and establish persistent infection in mammals