Analysis of the prevalence, secretion and function of a cell cycle-inhibiting factor in the melioidosis pathogen Burkholderia pseudomallei

Enteropathogenic and enterohaemorrhagic Escherichia coli express a cell cycle-inhibiting factor (Cif), that is injected into host cells via a Type III secretion system (T3SS) leading to arrest of cell division, delayed apoptosis and cytoskeletal rearrangements. A homologue of Cif has been identified in Burkholderia pseudomallei (CHBP; Cif homologue in B. pseudomallei; BPSS1385), which shares catalytic activity, but its prevalence, secretion and function are ill-defined. Among 43 available B. pseudomallei genome sequences, 33 genomes (76.7%) harbor the gene encoding CHBP. Western blot analysis using antiserum raised to a synthetic CHBP peptide detected CHBP in 46.6% (7/15) of clinical B. pseudomallei isolates from the endemic area. Secretion of CHBP into bacterial culture supernatant could not be detected under conditions where a known effector (BopE) was secreted in a manner dependent on the Bsa T3SS. In contrast, CHBP could be detected in U937 cells infected with B. pseudomallei by immunofluorescence microscopy and Western blotting in a manner dependent on bsaQ. Unlike E. coli Cif, CHBP was localized within the cytoplasm of B. pseudomallei-infected cells. A B. pseudomallei chbP insertion mutant showed a significant reduction in cytotoxicity and plaque formation compared to the wild-type strain that could be restored by plasmid-mediated trans-complementation. However, there was no defect in actin-based motility or multinucleated giant cell formation by the chbP mutant. The data suggest that the level or timing of CHBP secretion differs from a known Bsa-secreted effector and that CHBP is required for selected virulence-associated phenotypes in vitro

Regulation of Type VI Secretion System during Burkholderia pseudomallei Infection ▿

Type III and type VI secretion systems (T3SSs and T6SSs, respectively) are critical virulence determinants in several Gram-negative pathogens. In Burkholderia pseudomallei, the T3SS-3 and T6SS-1 clusters have been implicated in bacterial virulence in mammalian hosts. We recently discovered a regulatory cascade that coordinately controls the expression of T3SS-3 and T6SS-1. BsaN is a central regulator located within T3SS-3 for the expression of T3SS-3 effectors and regulators for T6SS-1 such as VirA-VirG (VirAG) and BprC. Whereas T6SS-1 gene expression was completely dependent on BprC when bacteria were grown in medium, the expression inside host cells was dependent on the two-component sensor-regulator VirAG, with the exception of the tssAB operon, which was dependent primarily on BprC. VirAG and BprC initiate different transcriptional start sites within T6SS-1, and VirAG is able to activate the hcp1 promoter directly. We also provided novel evidence that virAG, bprC, and tssAB are critical for T6SS-1 function in macrophages. Furthermore, virAG and bprC regulator mutants were avirulent in mice, demonstrating the absolute dependence of T6SS-1 expression on these regulators in vivo

Effects of circular myotomy on the healing of esophageal suture anastomosis: an experimental study Efeitos da miotomia circular na cicatrização das anastomoses esofágicas: estudo experimental

For esophageal reconstruction in newborns with esophageal atresia, esophageal reunion with an end-to-end anastomosis is the ideal procedure, although it may result in leaks and strictures due to tension on the suture line, mainly in cases with a wide gap between the ends. Circular myotomy (Livaditis' procedure) is the best method to elongate the proximal esophageal pouch and reduce anastomotic tension. This experimental investigation in dogs was undertaken to attempt to verify that circular myotomy decreases the anastomotic leak rate in newborns with wide gap esophageal atresia, and to analyze whether the technique promotes morphologic changes in the anastomotic scar. A pilot study demonstrated that it is necessary to resect more than 8 cm (40% of the total esophageal length) in order to obtain high leak rates. In the experimental project, such resection was performed in dogs divided into two groups (control group, anastomosis only, and experimental group, anastomosis plus circular myotomy in the proximal esophageal segment). The animals were killed in the 14th postoperative day, submitted to autopsy, and were evaluated as to the presence of leaks and strictures, as well as to the features (macroscopic and microscopic aspects) of the anastomosis. Leak rates were the same in both groups. Morphometric analysis revealed that in animals in the experimental group, the anastomotic scar was thinner than the control animals, and the isolated muscular manchette distal to the site of myotomy was replaced by fibrous tissue. Correspondingly, a decreased number of newly formed small vessels were noted in the experimental animals, compared to control animals. We concluded that circular myotomy does not decrease the incidence of anastomotic leaks, and it also promotes deleterious changes in anastomotic healing.<br>A anastomose término-terminal entre os segmentos esofágicos, embora constitua a melhor solução para a atresia do esôfago do recém-nascido, pode resultar em fístulas, deiscências ou estenoses devidas à tensão na linha de sutura, principalmente nos casos de grande distância entre os cotos. A miotomia circular do segmento proximal (manobra de Livaditis) é a técnica de alongamento esofágico mais utilizada. No presente trabalho experimental, realizado em cães, procurou-se verificar se esta técnica diminui o índice de deiscência de anastomose entre cotos sob grande tensão e estudar se ela promove alterações morfológicas na cicatriz da anastomose. O projeto piloto demonstrou que seria necessário ressecar 40% do esôfago para se obterem índices elevados de deiscência. No projeto experimental, realizou-se tal ressecção, e os cães foram divididos em dois grupos (grupo controle, apenas anastomose, e grupo experimental, anastomose com miotomia circular). Os animais foram mortos no 14º dia de pós-operatório e a seguir submetidos a necropsia, avaliando-se a presença de deiscência e estenose da anastomose. A avaliação microscópica constou de análise histológica qualitativa e histomorfometria da cicatriz da anastomose. Os índices de deiscência foram iguais nos dois grupos. Nos animais do grupo controle a espessura da fibrose e o número de vasos neoformados foram maiores que no grupo experimental. A avaliação morfométrica revelou que a miotomia promoveu diminuição na espessura da fibrose cicatricial. Concluiu-se que a miotomia circular não diminui a possibilidade de deiscência, além de promover alterações deletérias na cicatrização da anastomose

Melioidosis.

Burkholderia pseudomallei is a Gram-negative environmental bacterium and the aetiological agent of melioidosis, a life-threatening infection that is estimated to account for ∼89,000 deaths per year worldwide. Diabetes mellitus is a major risk factor for melioidosis, and the global diabetes pandemic could increase the number of fatalities caused by melioidosis. Melioidosis is endemic across tropical areas, especially in southeast Asia and northern Australia. Disease manifestations can range from acute septicaemia to chronic infection, as the facultative intracellular lifestyle and virulence factors of B. pseudomallei promote survival and persistence of the pathogen within a broad range of cells, and the bacteria can manipulate the host's immune responses and signalling pathways to escape surveillance. The majority of patients present with sepsis, but specific clinical presentations and their severity vary depending on the route of bacterial entry (skin penetration, inhalation or ingestion), host immune function and bacterial strain and load. Diagnosis is based on clinical and epidemiological features as well as bacterial culture. Treatment requires long-term intravenous and oral antibiotic courses. Delays in treatment due to difficulties in clinical recognition and laboratory diagnosis often lead to poor outcomes and mortality can exceed 40% in some regions. Research into B. pseudomallei is increasing, owing to the biothreat potential of this pathogen and increasing awareness of the disease and its burden; however, better diagnostic tests are needed to improve early confirmation of diagnosis, which would enable better therapeutic efficacy and survival