| Burkholderia pseudomallei is a notorious pathogen of human meloidosis with a high mortality and relapse rate, which has been endemic in Southeast Asia and Northern Australia. Recently, the first evidence-based predictive map of the global incidence and burden of melioidosis estimated 165,000(95% credible interval 68,000–412,000) human melioidosis cases per year worldwide and about 89,000(36,000–227,000) people death. B. pseudomallei has been designated a select agent(tier 1) by the US Centers for Disease Control and Prevention(CDC) since 2006 because of its potential as a biothreat organism, highlighting the importance of this emerging infectious disease. Clinical manifestations of melioidosis vary greatly from localized abscesses to severe pneumonia or life threatening sepsis with mortality up to 50% and relapse rate up to 20% regardless of standard antibiotics treatment. Diabetes is the strongest risk factor associated with melioidosis, with about 40-60% of melioidosis patients being diabetic. Not surprising, increasing numbers of research groups around the world are studying this versatile pathogen.China has been an epidemic focus and main region of melioidosis over decades, and almost all melioidosis cases were isolated or recorded in Hainan. However, the latest reprot of melioidosis in China have been dated back in the 90 th of centuray and the epidemic of melioidosis in China is urgent to be updated. Multilocus sequence typing(MLST) has been applied to the molecular epidemiological study of B. pseudomallei since 2003. The technique generates data that are easy-to-use, unambiguous, and readily comparable across laboratories. As no published study has yet applied MLST to B. pseudomallei isolates from Hainan we reasoned, given the prevalence of melioidosis in the province, it is urgent to investigate the epidemic and variation tendency of B. pseudomallei in China.Though B. pseudomallei was paid much attention overseas with annual incidence rates of up to 50 cases per 100,000 people(2012, N Engl J Med), it is not the case in China. Low awareness of the disease is mainly due to many reasons. The principal reason is the difficulty of diagnosis as the disease has a wide spectrum of clinical presentations, ranging from asymptomatic infection to disseminated septicaemic infection with a high misdiagnosis rate. Most doctors have not heard of this disease in local, not mention to those out of the island. We provided an analysis of melioidosis cases managed within Hainan province, China over a recent 11 years period.It was found in this restrospective study that there are local characteristics of melioidosis and the main headache for doctors in clinic was chronic infection and relapse. It has been reported that Burkholderia pseudomallei is able to invade host cells, and its capacity of persistence in cell can contribute to chronic infection of Burkholderia pseudomallei. Autophagy is thought to be a potent strategy of host cell to wipe out most intracellular bacteria by the autophagosomic-lysosomal degradation. Autophagosome can envelop bacteria and deliver them to lysosome for degradation. However, some pathogens have evolved smart strategies against the killing or trap of autophagy. Our previous study has discovered that Burkholderia pseudomallei can induce special mi RNAs to inhibit autophagosome formation and facilitate its survival in A549 cells. On the other hand, it has been reported that Burkholderia pseudomallei can induce MAP1LC3 B conversion in macrophages. However, little is unknown about the function of induced autophagosomes and how Burkholderia pseudomallei survive together with them which should be fatal to intercellular bacteria. Based on our latest expression array analysis in murine macrophages treated by B. pseudomallei, we found that there was more complex mechanism rather than simple inhibition on autophagosome formation. B. pseudomallei inhibited autophagy through the disruption of fusion of autophagosome and lysosome and this inhibition also associated with B. pseudomallei-derived mi RNA and miRNA-m RNA network regulation. This study raised the concerns about the importance of unobstructed autophagy for host to defense intercellular bacterial infection. Furthermore, it is suggested that microbial-derived-miRNAs-mediated regulation may be involved in more hidden pathogenesis of microbial infection and enlighten us a possibility of application of interference on specific mi RNA against bacterial infection.Methods1. A retrospective study of melioidosis in China1.1 A retrospective study design was employed. Data were collected from three major hospitals in Hainan province between 1 January 2002 and 22 April 2013.1.2 Suspected specimens were cultured on Columbia blood agar and identified with Bio Merieux system and 16 S DNA PCR.1.3 Analysis on risk factors, endemic tendency, geographic distribution, seasonal distribution and clinical characteristics.2. MLST analysis on B. pseudomallei2.1 Cases collection and bacterial strains: One hundred and two strains of B. pseudomallei were collected between December 2002 and December 2014.2.2 PCR and sequencing: PCR primers for amplification of seven housekeeping genes were modified and STs were defined and deposited in B. pseudomallei MLST database.2.3 e-BURST: The phylogenetic relationships of all strains were generated using e BURST v. 3 and UPGMA dendrogram was constructed based on the pair-wise differences.2.4 One case of melioidosis acquired by a traveler from Papua New Guinea.3. Autophagy inhibition by B. pseudomallei3.1 Plasmids construction for miR-Lipa-146 a and pcDNA3.1- Lipa.3.2 RNAi was used to evaluate the role of suspected genes of autophagy pathway.3.3 Confocal microscopy, super resolution imaging and TEM were applied to observe LC3 B conversion, cell fusion, intercellular bacteria and autolysosome or autolysosome formation.3.4 Survival of B. pseudomallei in macrophages was measured by CFU assay.3.5 qRT-PCR and western blot are for qualification of expressions.3.6 Luciferase assay is for verification of the mi R-146 a targeting on Lipa mRNA.3.7 Statistical analysisResults1. A retrospective study of melioidosis in China1.1 There was an increasing trend in the incidence of melioidosis in China.1.2 There were characteristics about melioidosis in China, different from other places.1.3 melioidosis outbreak associated with the immunity status of host and represented as chronic melioidosis infection.2. MLST analysis2.1. One hundred and two strains were characterized by MLST and resolved into 41 sequence types(ST), including eight novel STs, ST-1341 and from ST-1345 to ST-1351.2.2. ST-46, ST-50, ST-55, ST-58, ST-70 and ST-1095 were predominant, and the strains belonging to these STs were first isolated from China and Thailand, an evolutionary tree and e-BURST graphics suggesting a relatively close link between these two endemic focus.2.3. The characteristics of alleles in B. pseudomallei strains we studied were analyzed.,gmh D showed a high diversity among B. pseudomallei strains in China.3. Burkholderia pseudomallei-derived miR-146 a disrupts autophagy and promotes bacterial survival in murine macrophage.3.1 B. pseudomallei can increase autophagosome generation and coexist with it.3.2 Autophagosome can only engulf and restrict B. pseudomallei at early phase of infection.3.3 Autophagy was blocked by B. pseudomallei in macrophages.3.4 Depression of Lipa could contribute to the block of autophagy in macrophages and Lipa is the important protein of autolysosome formation.3.5 miR-146 a was upregulated after B. pseudomallei-infection.3.6 miR-146 a targets Lipa.3.7 miR-146a-Lipa regulated the fusion of autophagosome and lysosome and controls the survival of intercellular B. pseudomallei.ConclusionsThe research results on the one hand enhance our clinical recognition of melioidosis and understand the epidemic situation of melioidosis and variation characteristic, perfected the Burkholderia pseudomallei strain genetic information databases, can provides reference to diagnosis of melioidosis for clinicians especially medical workers from epidemic areas, and provide guidance for traceability and early warning of melioidosis in china; on the other hand, through in-depth study of Burkholderia pseudomallei escape mechanism of autophagy, provides a mechanism to explain clinical problems of melioidosis(infection of chronic and relapse), these findings suggests that the important role of mi RNA in the host cell signaling pathway induced by microorganisms, provide a new way of thinking for clinical treatment against melioidosis infection. |
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