Huimin Zhang

Streptococcus suis infection: An emerging/reemerging challenge of bacterial infectious diseases?

Streptococcus suis (S. suis) is a family of pathogenic gram-positive bacterial strains that represents a primary health problem in the swine industry worldwide. S. suis is also an emerging zoonotic pathogen that causes severe human infections clinically featuring with varied diseases/syndromes (such as meningitis, septicemia, and arthritis). Over the past few decades, continued efforts have made significant progress toward better understanding this zoonotic infectious entity, contributing in part to the elucidation of the molecular mechanism underlying its high pathogenicity. This review is aimed at presenting an updated overview of this pathogen from the perspective of molecular epidemiology, clinical diagnosis and typing, virulence mechanism, and protective antigens contributing to its zoonosis.

Attenuation of Streptococcus suis virulence by the alteration of bacterial surface architecture

NeuB, a sialic acid synthase catalyzes the last committed step of the de novo biosynthetic pathway of sialic acid, a major element of bacterial surface structure. Here we report a functional NeuB homologue of Streptococcus suis, a zoonotic agent, and systematically address its molecular and immunological role in bacterial virulence. Disruption of neuB led to thinner capsules and more susceptibility to pH, and cps2B inactivation resulted in complete absence of capsular polysaccharides. These two mutants both exhibited increased adhesion and invasion to Hep-2 cells and improved sensibility to phagocytosis. Not only do they retain the capability of inducing the release of host pro-inflammatory cytokines, but also result in the faster secretion of IL-8. Easier cleaning up of the mutant strains in whole blood is consistent with virulence attenuation seen with experimental infections of both mice and SPF-piglets. Therefore we concluded that altered architecture of S. suis surface attenuates its virulence.

Genomic insights into the ESBL and MCR-1-producing ST648 Escherichia coli with multi-drug resistance

Polymyxin acts as an ultimate line of refuge against the severe infections by multidrug-resistant Gram-negative pathogens. This conventional idea is challenged dramatically by the recent discovery of mobile colistin resistance gene (mcr-1) is prevalent in food animals and human beings worldwide. More importantly, the mcr-1 gene was found to be co-localized with other antibiotic resistance genes, raising the possibility that super-bugs with pan-drug resistance are emerging. However, little is reported on the genomes of the mcr-1-positive bacterial host reservoirs. Here we report genome sequencing of three human isolates of the mcr-1-positive Escherichia coli (E15004, E15015 and E15017) and define general features through analyses of bacterial comparative genomics. Further genomic mining together with sequence typing allowed us to elucidate that the MCR-1-carrying E. coli E15017 belongs to the sequence type ST648 and coproduces extended-spectrum β-lactamase (ESBL). Given the fact that ST648 has been known to associate with either New Delhi metallo-β-lactamase 1 or ESBL, our results highlighted the possibility of ST648 as an epidemic clone with multidrug resistances.摘要通常认为多粘菌素是抵御多药耐受的革兰阴性病原菌严重感染的最后一道防线。 最新研究表明在动物类食品和人体肠道菌群中存在一种多粘菌素抗性基因 (mcr-1)。这一惊人发现对上述传统观点提出了严重挑战。更为惊讶的是, 该 mcr-1 基因可与其他抗生素抗性基因共存, 极大增加了产生具有广泛耐药的超级 细菌的可能性。但是, 关于mcr-1 阳性细菌的基因组学特征鲜见报道。本研究对 3 株 mcr-1 阳性人源大肠杆菌(E15004, E15015 和E15017)开展了全基因组 测序, 并通过比较基因组学描述了其基因组学特征。基因组深入挖掘和序列分型 实验揭示了序列型为ST648 的大肠杆菌E15017 不仅编码MCR-1 多粘菌素耐药 基因, 而且携带超广谱β-内酰胺耐药基因(ESBL)。鉴于序列型ST648 的大肠杆 菌通常与新德里β-内酰胺耐药-1 (NDM-1) 或ESBL 紧密关联这一事实, 我们的 研究结果提示了具有MCR-1 和ESBL 耐药的ST648 细菌发展成流行性克隆的潜 在风险。

Mycobacterium smegmatis BioQ defines a new regulatory network for biotin metabolism.

Biotin (vitamin H), the sulfur‐containing enzyme cofactor, is an essential micronutrient for three domains of life. Given the fact that biotin is an energetically expensive molecule whose de novo biosynthesis demands 20 ATP equivalents each, it is reasonable that bacteria have evolved diversified mechanisms in various microorganisms to tightly control biotin metabolism. Unlike the Escherichia coli BirA, the prototypical bi‐functional version of biotin protein ligase (BPL) in that it acts as a repressor for biotin biosynthesis pathway, the BirA protein of Mycobacterium smegmatis (M. smegmatis), a closely relative of the tuberculosis‐causing pathogen, Mycobacterium tuberculosis, lacked the DNA‐binding activity. It raised a possibility that an alternative new regulator might be present to compensate the loss of regulatory function. Here we report that this is the case. Genomic context analyses of M. smegmatis detected a newly identified BioQ homolog classified into the TetR family of transcription factor and its recognizable palindromes. The M. smegmatis BioQ protein was overexpressed and purified to homogeneity. Size‐exclusion chromatography combined with chemical cross‐linking studies demonstrated that the BioQ protein had a propensity to dimerize. The promoters of bioFD and bioQ/B were mapped using 5′‐RACE. Electrophoretic mobility shift assays revealed that BioQ binds specifically to the promoter regions of bioFD and bioQ/B. Further DNase I foot‐printing elucidated the BioQ‐binding palindromes. Site‐directed mutagenesis suggested the important residues critical for BioQ/DNA binding. The isogenic mutant of bioQ (ΔbioQ) was generated using the approach of homologous recombination. The in vivo data from the real‐time qPCR combined with the lacZ transcriptional fusion experiments proved that removal of bioQ gave significant increment with expression of bio operons. Also, expression of bio operons were repressed by exogenous addition of biotin, and this repression seemed to depend on the presence of BioQ protein. Thereby, we believed that M. smegmatis BioQ is not only a negative auto‐regulator but also a repressor for bioFD and bioB operons involved in the biotin biosynthesis pathway. Collectively, this finding defined the two‐protein paradigm of BirA and BioQ, representing a new mechanism for bacterial biotin metabolism.

Brucella BioR Regulator Defines a Complex Regulatory Mechanism for Bacterial Biotin Metabolism

The enzyme cofactor biotin (vitamin H or B7) is an energetically expensive molecule whose de novo biosynthesis requires 20 ATP equivalents. It seems quite likely that diverse mechanisms have evolved to tightly regulate its biosynthesis. Unlike the model regulator BirA, a bifunctional biotin protein ligase with the capability of repressing the biotin biosynthetic pathway, BioR has been recently reported by us as an alternative machinery and a new type of GntR family transcriptional factor that can repress the expression of the bioBFDAZ operon in the plant pathogen Agrobacterium tumefaciens. However, quite unusually, a closely related human pathogen, Brucella melitensis, has four putative BioR-binding sites (both bioR and bioY possess one site in the promoter region, whereas the bioBFDAZ [bio] operon contains two tandem BioR boxes). This raised the question of whether BioR mediates the complex regulatory network of biotin metabolism. Here, we report that this is the case. The B. melitensis BioR ortholog was overexpressed and purified to homogeneity, and its solution structure was found to be dimeric. Functional complementation in a bioR isogenic mutant of A. tumefaciens elucidated that Brucella BioR is a functional repressor. Electrophoretic mobility shift assays demonstrated that the four predicted BioR sites of Brucella plus the BioR site of A. tumefaciens can all interact with the Brucella BioR protein. In a reporter strain that we developed on the basis of a double mutant of A. tumefaciens (the ΔbioR ΔbioBFDA mutant), the β-galactosidase (β-Gal) activity of three plasmid-borne transcriptional fusions (bioBbme-lacZ, bioYbme-lacZ, and bioRbme-lacZ) was dramatically decreased upon overexpression of Brucella bioR. Real-time quantitative PCR analyses showed that the expression of bioBFDA and bioY is significantly elevated upon removal of bioR from B. melitensis. Together, we conclude that Brucella BioR is not only a negative autoregulator but also a repressor of expression of bioY and bio operons that separately function in biotin transport and the biosynthesis pathway.

Two novel regulators of N‐acetyl‐galactosamine utilization pathway and distinct roles in bacterial infections

Bacterial pathogens can exploit metabolic pathways to facilitate their successful infection cycles, but little is known about roles of d‐galactosamine (GalN)/N‐acetyl‐d‐galactosamine (GalNAc) catabolism pathway in bacterial pathogenesis. Here, we report the genomic reconstruction of GalN/GalNAc utilization pathway in Streptococci and the diversified aga regulons. We delineated two new paralogous AgaR regulators for the GalN/GalNAc catabolism pathway. The electrophoretic mobility shift assays experiment demonstrated that AgaR2 (AgaR1) binds the predicted palindromes, and the combined in vivo data from reverse transcription quantitative polymerase chain reaction and RNA‐seq suggested that AgaR2 (not AgaR1) can effectively repress the transcription of the target genes. Removal of agaR2 (not agaR1) from Streptococcus suis 05ZYH33 augments significantly the abilities of both adherence to Hep‐2 cells and anti‐phagocytosis against RAW264.7 macrophage. As anticipated, the dysfunction in AgaR2‐mediated regulation of S. suis impairs its pathogenicity in experimental models of both mice and piglets. Our finding discovered two novel regulators specific for GalN/GalNAc catabolism and assigned them distinct roles into bacterial infections. To the best of our knowledge, it might represent a first paradigm that links the GalN/GalNAc catabolism pathway to bacterial pathogenesis.

Paracoccus denitrificans possesses two BioR homologs having a role in regulation of biotin metabolism.

Recently, we determined that BioR, the GntR family of transcription factor, acts as a repressor for biotin metabolism exclusively distributed in certain species of α‐proteobacteria, including the zoonotic agent Brucella melitensis and the plant pathogen Agrobacterium tumefaciens. However, the scenario is unusual in Paracoccus denitrificans, another closely related member of the same phylum α‐proteobacteria featuring with denitrification. Not only does it encode two BioR homologs Pden_1431 and Pden_2922 (designated as BioR1 and BioR2, respectively), but also has six predictive BioR‐recognizable sites (the two bioR homolog each has one site, whereas the two bio operons (bioBFDAGC and bioYB) each contains two tandem BioR boxes). It raised the possibility that unexpected complexity is present in BioR‐mediated biotin regulation. Here we report that this is the case. The identity of the purified BioR proteins (BioR1 and BioR2) was confirmed with LC‐QToF‐MS. Phylogenetic analyses combined with GC percentage raised a possibility that the bioR2 gene might be acquired by horizontal gene transfer. Gel shift assays revealed that the predicted BioR‐binding sites are functional for the two BioR homologs, in much similarity to the scenario seen with the BioR site of A. tumefaciens bioBFDAZ. Using the A. tumefaciens reporter system carrying a plasmid‐borne LacZ fusion, we revealed that the two homologs of P. denitrificans BioR are functional repressors for biotin metabolism. As anticipated, not only does the addition of exogenous biotin stimulate efficiently the expression of bioYB operon encoding biotin transport/uptake system BioY, but also inhibits the transcription of the bioBFDAGC operon resembling the de novo biotin synthetic pathway. EMSA‐based screening failed to demonstrate that the biotin‐related metabolite is involved in BioR‐DNA interplay, which is consistent with our former observation with Brucella BioR. Our finding defined a complex regulatory network for biotin metabolism in P. denitrificans by two BioR proteins.

Preplasma effects on the generation of high-energy protons in ultraintense laser interaction with foil targets

It is shown that the intense quasistatic electric and magnetic fields self-generated near the axis of the laser-driven channel in an appropriately profiled preplasma during ultraintense laser interaction with a thin target can create dense relativistic electron bunches. The latter easily penetrate through the target and can greatly enhance the sheath field at the rear, resulting in significant increase in the laser-to-ion energy conversion efficiency and the maximum energy of the target normal sheath accelerated ions. Particle-in-cell simulations show that with a hydrogen targets a proton beam of peak energy ∼38 MeV and energy conversion efficiency ≥6.5% can be produced by a linearly polarized 5 × 1019 W/cm2 laser. An analytical model is also proposed and its results agree well with those of the simulations.

Functional definition of BirA suggests a biotin utilization pathway in the zoonotic pathogen Streptococcus suis.

Biotin protein ligase is universal in three domains of life. The paradigm version of BPL is the Escherichia coli BirA that is also a repressor for the biotin biosynthesis pathway. Streptococcus suis, a leading bacterial agent for swine diseases, seems to be an increasingly-important opportunistic human pathogen. Unlike the scenario in E. coli, S. suis lacks the de novo biotin biosynthesis pathway. In contrast, it retains a bioY, a biotin transporter-encoding gene, indicating an alternative survival strategy for S. suis to scavenge biotin from its inhabiting niche. Here we report functional definition of S. suis birA homologue. The in vivo functions of the birA paralogue with only 23.6% identity to the counterpart of E. coli, was judged by its ability to complement the conditional lethal mutants of E. coli birA. The recombinant BirA protein of S. suis was overexpressed in E. coli, purified to homogeneity and verified with MS. Both cellulose TLC and MALDI-TOFF-MS assays demonstrated that the S. suis BirA protein catalyzed the biotinylation reaction of its acceptor biotin carboxyl carrier protein. EMSA assays confirmed binding of the bioY gene to the S. suis BirA. The data defined the first example of the bifunctional BirA ligase/repressor in Streptococcus.

Binding of Shewanella FadR to the fabA fatty acid biosynthetic gene: implications for contraction of the fad regulon.

ABSTRACTThe Escherichia colifadR protein product, a paradigm/prototypical FadR regulator, positively regulates fabA and fabB, the two critical genes for unsaturated fatty acid (UFA) biosynthesis. However the scenario in the other Ɣ–proteobacteria, such as Shewanella with the marine origin, is unusual in that Rodionov and coworkers predicted that only fabA (not fabB) has a binding site for FadR protein. It raised the possibility of fad regulon contraction. Here we report that this is the case. Sequence alignment of the FadR homologs revealed that the N-terminal DNA-binding domain exhibited remarkable similarity, whereas the ligand-accepting motif at C-terminus is relatively-less conserved. The FadR homologue of S. oneidensis (referred to FadR_she) was over-expressed and purified to homogeneity. Integrative evidence obtained by FPLC (fast protein liquid chromatography) and chemical cross-linking analyses elucidated that FadR_she protein can dimerize in solution, whose identity was determined by MALDI-TOF-MS. In vitro data from electrophoretic mobility shift assays suggested that FadR_she is almost functionally-exchangeable/equivalent to E. coli FadR (FadR_ec) in the ability of binding the E. coli fabA (and fabB) promoters. In an agreement with that of E. coli fabA, S. oneidensisfabA promoter bound both FadR_she and FadR_ec, and was disassociated specifically with the FadR regulatory protein upon the addition of long-chain acyl-CoA thioesters. To monitor in vivo effect exerted by FadR on Shewanella fabA expression, the native promoter of S. oneidensisfabA was fused to a LacZ reporter gene to engineer a chromosome fabA-lacZ transcriptional fusion in E. coli. As anticipated, the removal of fadR gene gave about 2-fold decrement of ShewanellafabA expression by β-gal activity, which is almost identical to the inhibitory level by the addition of oleate. Therefore, we concluded that fabA is contracted to be the only one member of fad regulon in the context of fatty acid synthesis in the marine bacteria Shewanella genus.