Xitong Yuan

Comparative Proteomics Analyses Reveal the virB of B. melitensis Affects Expression of Intracellular Survival Related Proteins

Backgound Brucella melitensis is a facultative, intracellular, pathogenic bacterium that replicates within macrophages. The type IV secretion system encoded by the virB operon (virB) is involved in Brucella intracellular survival. However, the underlying molecular mechanisms, especially the target proteins affected by the virB, remain largely unclear. Methodology/Principal Findings In order to define the proteins affected by virB, the proteomes of wild-type and the virB mutant were compared under in vitro conditions where virB was highly activated. The differentially expressed proteins were identified by MALDI-TOF-MS. Forty-four down-regulated and eighteen up-regulated proteins which exhibited a 2-fold or greater change were identified. These proteins included those involved in amino acid transport and metabolism, lipid metabolism, energy production, cell membrane biogenesis, translation, post-translational modifications and protein turnover, as well as unknown proteins. Interestingly, several important virulence related proteins involved in intracellular survival, including VjbR, DnaK, HtrA, Omp25, and GntR, were down-regulated in the virB mutant. Transcription analysis of virB and vjbR at different growth phase showed that virB positively affect transcription of vjbR in a growth phase dependent manner. Quantitative RT-PCR showed that transcription of these genes was also affected by virB during macrophage cell infection, consistent with the observed decreased survival of the virB mutant in macrophage. Conclusions/Significance These data indicated that the virB operon may control the intracellular survival of Brucella by affecting the expression of relevant proteins.

Impact of Hfq on Global Gene Expression and Intracellular Survival in Brucella melitensis

Brucella melitensis is a facultative intracellular bacterium that replicates within macrophages. The ability of brucellae to survive and multiply in the hostile environment of host macrophages is essential to its virulence. The RNA-binding protein Hfq is a global regulator that is involved in stress resistance and pathogenicity. Here we demonstrate that Hfq is essential for stress adaptation and intracellular survival in B. melitensis. A B. melitensis hfq deletion mutant exhibits reduced survival under environmental stresses and is attenuated in cultured macrophages and mice. Microarray-based transcriptome analyses revealed that 359 genes involved in numerous cellular processes were dysregulated in the hfq mutant. From these same samples the proteins were also prepared for proteomic analysis to directly identify Hfq-regulated proteins. Fifty-five proteins with significantly affected expression were identified in the hfq mutant. Our results demonstrate that Hfq regulates many genes and/or proteins involved in metabolism, virulence, and stress responses, including those potentially involved in the adaptation of Brucella to the oxidative, acid, heat stress, and antibacterial peptides encountered within the host. The dysregulation of such genes and/or proteins could contribute to the attenuated hfq mutant phenotype. These findings highlight the involvement of Hfq as a key regulator of Brucella gene expression and facilitate our understanding of the role of Hfq in environmental stress adaptation and intracellular survival of B. melitensis.

Identification of a Novel Small Non-Coding RNA Modulating the Intracellular Survival of Brucella melitensis.

Bacterial small non-coding RNAs (sRNAs) are gene expression modulators respond to environmental changes, stressful conditions, and pathogenesis. In this study, by using a combined bioinformatic and experimental approach, eight novel sRNA genes were identified in intracellular pathogen Brucella melitensis. BSR0602, one sRNA that was highly induced in stationary phase, was further examined and found to modulate the intracellular survival of B. melitensis. BSR0602 was present at very high levels in vitro under stresses similar to those encountered during infection in host macrophages. Furthermore, BSR0602 was found to be highly expressed in the spleens of infected mice, suggesting its potential role in the control of pathogenesis. BSR0602 targets the mRNAs coding for gntR, a global transcriptional regulator, which is required for B. melitensis virulence. Overexpression of BSR0602 results in distinct reduction in the gntR mRNA level. B. melitensis with high level of BSR0602 is defective in bacteria intracellular survival in macrophages and defective in growth in the spleens of infected mice. Therefore, BSR0602 may directly inhibit the expression of gntR, which then impairs Brucellae intracellular survival and contributes to Brucella infection. Our findings suggest that BSR0602 is responsible for bacterial adaptation to stress conditions and thus modulate B. melitensis intracellular survival.

Human brucellosis, a heterogeneously distributed, delayed, and misdiagnosed disease in china.

TO THE EDITOR—Brucellosis is a common zoonotic disease worldwide [1]. The epidemiology of human brucellosis has drastically changed owing to changing sanitary and socioeconomic conditions, and the substantial increase in international travel. New foci of human brucellosis have emerged or reemerged, with brucellosis becoming a travel-related disease, particularly in nonendemic areas [2–4]. Awareness of the geographic distribution and characteristics of brucellosis is valuable not only for the disease control in endemic areas, but also for preventing and diagnosing travel-associated disease in nonendemic areas [5, 6]. To investigate the geographic distribution of the human brucellosis epidemic in China, data were extracted from the National Notifiable Disease Surveillance System and analyzed. From December 2004 to July 2010, 141 604 laboratoryconfirmed cases were reported. A geographic distribution analysis showed that brucellosis is mainly distributed in some of the northern provinces of China. The 5 provinces with the highest incidence include Inner Mongolia (45.83%), Shanxi (13.94%), Heilongjiang (13.82%), Jilin (10.29%), and Hebei (6.31%); these provinces accounted for >90% of the reported cases. Nationwide, the annual incidence, at a county level, ranged from 0.00 to 1395.84 per 100 000 (Figure 1). Timely diagnosis is important for treating brucellosis and preventing chronic infections [7]. We examined the case data for possible contributing factors to delayed diagnoses. Of these 141 604 confirmed cases, only 26.98% were diagnosed within 7 days of symptom onset, 43.83% within 14 days, and 2.39% longer than 6 months, indicating that a large proportion of the cases had a delayed diagnosis. Further analysis showed that the average diagnosis delay was shorter in the high-incidence areas than in the low-incidence areas (35 vs 59 days, P < .001), and shorter in urban areas than in villages (32.55 vs 40.62 days, P < .001). In addition, patients who traveled or migrated across provinces had longer delays than did patients residing within an area (43.54 vs 36.82 days, P < .001). These data indicated that diagnosis delays were multifactorial and may have contributed to the chronicity of brucellosis in some population segments. Epidemiological information regarding 2060 cases were collected from brucellosis clinics and analyzed to investigate the reasons for the delays in diagnosis. Surprisingly, 57.62% of the patients had been misdiagnosed or suspected of having other diseases with similar clinical symptoms. Approximately 24.34% of the “delayed diagnosis” patients had traveled to or migrated from a high-risk area. All the patients had experienceddirect contactwith animals and/or consumed contaminated animal products prior to disease onset. The absence of characteristic symptoms and a general unawareness of epidemiological information contributed to the misdiagnoses. The lackof typical clinicalmanifestations makes it difficult to diagnose brucellosis, primarily contributing to the misdiagnosis and delay. The availability of additional information regarding the geographic

High Severity and Fatality of Human Infections With Avian Influenza A(H7N9) Infection in China

Severe infection by a novel influenza virus, distinct from the circulating human influenza A virus, in humans usually heralds a sporadic pattern of severehuman infection or an influenza pandemic [1]. Accordingly, the discovery of the novel avian influenza A(H7N9) virus is of great public health significance [2]. Because this virus has not been detected previously in humans or in animals, many urgent questions and global public health concerns need to be addressed [3]. Study of the early cases could provide key

Asymptomatic brucellosis infection in humans: implications for diagnosis and prevention

Human brucellosis is mainly caused by contact with Brucella-infected animals and their secretions and carcasses. Individuals who are continuously in contact with animals are considered to be at a high risk but only some show symptoms and are diagnosed as cases of brucellosis. Here, we showed that asymptomatic brucellosis infections occur among humans. Asymptomatic infections mainly result from less frequent contact with Brucella and/or contact with low-virulence Brucella. In our study, patients with asymptomatic infection had low antibody titres and different contact patterns. Awareness of asymptomatic infection is important for early diagnosis of brucellosis and prevention of chronic infection.

Changes of predominant species/biovars and sequence types of Brucella isolates, Inner Mongolia, China

BackgroundHuman brucellosis incidence in China was divided into 3 stages, high incidence (1950-1960s), decline (1970-1980s) and re-emergence (1990-2000s). Human brucellosis has been reported in all the 32 provinces, of which Inner Mongolia has the highest prevalence, accounting for over 40% of the cases in China. To investigate the etiology alteration of human brucellosis in Inner Mongolia, the species, biovars and genotypes of 60 Brucella isolates from this province were analyzed.MethodsSpecies and biovars of the Brucella strains isolated from outbreaks were determined based on classical identification procedures. Strains were genotyped by multi locus sequence typing (MLST). Sequences of 9 housekeeping genes were obtained and sequence types were defined. The distribution of species, biovars and sequence types (STs) among the three incidence stages were analyzed and compared.ResultsThe three stages of high incidence, decline and re-emergence were predominated by B. melitensis biovar 2 and 3, B. abortus biovar 3, and B. melitensis biovar 1, respectively, implying changes in the predominant biovars. Genotyping by MLST revealed a total of 14 STs. Nine STs (from ST28 to ST36), accounting for 64.3% of all the STs, were newly defined and different from those observed in other countries. Different STs were distributed among the three stages. ST8 was the most common ST in 1950-1960s and 1990-2000s, while ST2 was the most common in 1970-1980s.ConclusionsThe prevalence of biovars and sequence types of Brucella strains from Inner Mongolia has changed over time in the three stages. Compared with those from other countries, new sequence types of Brucella strains exist in China.

Vaccination with recombinant flagellar proteins FlgJ and FliN induce protection against Brucella abortus 544 infection in BALB/c mice

Brucella has been considered as a non-motile, facultative intracellular pathogenic bacterium. However, the genome sequences of different Brucella species reveal the presence of the flagellar genes needed for the construction of a functional flagellum. Due to its roles in the interaction between pathogen and host, we hypothesized that some of the flagellar proteins might induce protective immune responses and these proteins will be good subunit vaccine candidates. This study was conducted to screening of protective antigens among these flagellar proteins. Firstly, according to the putative functional roles, a total of 30 flagellar genes of Brucella abortus were selected for in vitro expression. 15 of these flagellar genes were successfully expressed as his-tagged recombinant proteins in Escherichia coli ER2566. Then, these proteins were purified and used to analyze their T cell immunity induction activity by an in vitro gamma interferon (IFN-γ) assay. Five of the flagellar proteins could stimulate significantly higher levels of IFN-γ secretion in splenocytes from S19 immunized mice, indicating their T cell induction activity. Finally, immunogenicity and protection activity of these 5 flagellar proteins were evaluated in BALB/c mice. Results showed that immunization with FlgJ (BAB1_0260) or FliN (BAB2_0122) plus adjuvant could provide protection against B. abortus 544 infection. Furthermore, mice immunized with FlgJ and FliN developed a vigorous immunoglobulin G response, and in vitro stimulation of their splenocytes with immunizing proteins induced the secretion of IFN-γ. Altogether, these data suggest that flagellar proteins FlgJ and FliN are protective antigens that could produce humoral and cell-mediated responses in mice and candidates for use in future studies of vaccination against brucellosis.

Development of an extended multilocus sequence typing for genotyping of Brucella isolates

By amplifying and sequencing longer sequences, an extended multi locus sequence typing (EMLST) theme was developed for Brucella. 61 isolates were genotyped by the EMLST with increased resolution. This strategy could be extended to other bacteria to improve MLST genotyping resolution without additional loci.

Genome Sequences of Brucella melitensis 16M and Its Two Derivatives 16M1w and 16M13w, Which Evolved In Vivo

Brucella melitensis is an intracellular pathogen that induces chronic infection in humans. Here, we report the genome sequences of 16M and its two derivatives, 16M1w and 16M13w, which were allowed to adapt in vivo for 1 and 13 weeks, respectively. Our findings contribute to the investigation of adaptive mutations and mechanisms of chronic infection by B. melitensis.