Genomotyping of Coxiella burnetii using microarrays reveals a conserved genomotype for hard tick isolates

C. burnetii is a Gram-negative intracellular Y-proteobacteria that causes the zoonotic disease Q fever. Q fever can manifest as an acute or chronic illness. Different typing methods have been previously developed to classify C. burnetii isolates to explore its pathogenicity. Here, we report a comprehensive genomotyping method based on the presence or absence of genes using microarrays. The genomotyping method was then tested in 52 isolates obtained from different geographic areas, different hosts and patients with different clinical manifestations. The analysis revealed the presence of 10 genomotypes organized into 3 groups, with a topology congruent with that obtained through multi-spacer typing. We also found that only 4 genomotypes were specifically associated with acute Q fever, whereas all of the genomotypes could be associated to chronic human infection. Serendipitously, the genomotyping results revealed that all hard tick isolates, including the Nine Mile strain, belong to the same genomotype.     

Role of antigens and virulence factors of Salmonella enterica serovar Typhi in its pathogenesis

Salmonella enterica serovar Typhi (S. Typhi), the aetiologic agent of typhoid fever, is a human restricted pathogen. The molecular mechanism of Salmonella pathogenicity is complex. The investigations of the molecular mechanisms of Salmonella virulence factors have shown that pathogenic Salmonella spp. are distinguished from their non-pathogenic relatives by the presence of specific pathogenicity genes, often organized in so-called pathogenicity islands (PIs). The type III secretion system (T3SS) proteins encoded by two Salmonella PIs (SPIs) are associated with the pathogenicity at molecular level. The identification of T3SS has provided new insight into the molecular factors and mechanisms underlying bacterial pathogenesis. The T3SS encoded by SPI-1 contains invasion genes; while SPI-2 is responsible for intracellular pathogenesis and has a crucial role for systemic S. enterica infections. These studies reveal a complex set of pathogenic interferences between intracellular Salmonella and its host cells. The understanding of the mechanisms by which Salmonella evade the host defense system and establish pathogenesis will be important for proper disease management.     

Coxiella burnetii survival in THP-1 monocytes involves the impairment of phagosome maturation: IFN-gamma mediates its restoration and bacterial killing

The subversion of microbicidal functions of macrophages by intracellular pathogens is critical for their survival and pathogenicity. The replication of Coxiella burnetii, the agent of Q fever, in acidic phagolysosomes of nonphagocytic cells has been considered as a paradigm of intracellular life of bacteria. We show in this study that C. burnetii survival in THP-1 monocytes was not related to phagosomal pH because bacterial vacuoles were acidic independently of C. burnetii virulence. In contrast, virulent C. burnetii escapes killing in resting THP-1 cells by preventing phagosome maturation. Indeed, C. burnetii vacuoles did not fuse with lysosomes because they were devoid of cathepsin D, and did not accumulate lysosomal trackers; the acquisition of markers of late endosomes and late endosomes-early lysosomes was conserved. In contrast, avirulent variants of C. burnetii were eliminated by monocytes and their vacuoles accumulated late endosomal and lysosomal markers. The fate of virulent C. burnetii in THP-1 monocytes depends on cell activation. Monocyte activation by IFN-gamma restored C. burnetii killing and phagosome maturation as assessed by colocalization of C. burnetii with active cathepsin D. In addition, when IFN-gamma was added before cell infection, it was able to stimulate C. burnetii killing but it also induced vacuolar alkalinization. These findings suggest that IFN-gamma mediates C. burnetii killing via two distinct mechanisms, phagosome maturation, and phagosome alkalinization. Thus, the tuning of vacuole biogenesis is likely a key part of C. burnetii survival and the pathophysiology of Q fever.     

Backbone and side-chain 1H, 13C and 15N assignments of the PPIase domain of macrophage infectivity potentiator (Mip) protein from Coxiella burnetii

Coxiella burnetii is an obligate intracellular gram-negative bacterium uniquely evolved to thrive in the inhospitable phagolysosome of macrophage. C. burnetii causes Q fever in humans and animals, which is emerging as a global public health concern. It is highly infectious and designated as a category B biowarfare agent because of its ubiquitous nature, abundant natural reservoirs, high resistance to environmental conditions, ease of transmission and low infectious dose. The lack of knowledge and awareness of C. burnetii leads to under-reporting and under-diagnosing of Q fever cases. Therefore, further understanding of the interactions between the infected host and the bacteria is necessary. C. burnetii macrophage infectivity potentiator (cb-Mip) is a secreted protein of 230 amino acids involving in intracellular survival of the pathogen. cb-Mip belongs to the family of FK506 binding protein, which possesses peptidyl-prolyl cis/trans isomerase (PPIase) activity. Besides acting as a PPIase, Mip protein homolog has been identified as virulence factor of many intracellular pathogenic microorganisms. In the present study, we report the near complete resonance assignments of the PPIase domain-containing region of Mip protein of C. burnetii. Secondary structure prediction based on chemical shift index analysis indicates that the protein adopts a predominately beta-strand structure, which is consistent with the crystal structure of homologous Mip protein in Legionella pneumophila.     

Asymmetric adaptation to indolic and aliphatic glucosinolates in the B and Q sibling species of Bemisia tabaci (Hemiptera: Aleyrodidae)

The role glucosinolates play in defending plants against phloem feeders such as aphids and whiteflies is currently not clear as these herbivores may avoid bringing glucosinolates from the phloem sap into contact with myrosinase enzymes. Here, we investigated the effects of high levels of aliphatic and indolic glucosinolates on life history traits and detoxification gene expression in two sibling species, B and Q, of the whitefly Bemisia tabaci. High levels of aliphatic glucosinolates decreased the average oviposition rate of both species and reduced the survival and developmental rate of Q nymphs. High levels of indolic glucosinolates decreased the oviposition rate and survival of nymphal stages of the B species and the developmental rate of both species. Molecular analyses revealed two major asymmetries between the B and Q species. First, specific GST genes (BtGST1 and BtGST2) were significantly induced during exposure to indolic glucosinolates only in Q. This may reflect the genes putative involvement in indolic glucosinolates detoxification and explain the species' good performance on plants accumulating indolic glucosinolates. Second, the constitutive expression of eight of the 10 detoxification genes analysed was higher in the Q species than in the B species. Interestingly, four of these genes were induced in B in response to high levels of glucosinolates. It seems, therefore, that the B and Q species differ in their 'optimal defence strategy'. B utilizes inducible defences that are profitable if the probability of experiencing the stress is small and its severity is low, while Q invests significant resources in being always 'ready' for a challenge.     

Stress-inducible bacterial proteins and virulence

Different species of pathogenic bacteria, including Salmonella, Neisseria, Listeria and Francisella have been used to demonstrate relationship between the synthesis of stressor induced proteins by cells and the phenotypic manifestation of their virulence. The impact of such external factors as high temperature, low pH, osmolarity, substrate limitation, the content of active forms of oxygen, etc. is accompanied by the synthesis of different stressor induced proteins playing a complex role. Under unfavorable environmental conditions the synthesis of these proteins ensures the survival of the infective agents. Under conditions of a macroorganism synthesis of some stressor induced proteins promotes the survival of infective agents and their resistance to the action of humoral and cell-mediated protective factors of the host. As is known, the expression of virulence genes is not constitutive. The expression of these genes greatly depends on environmental conditions and its induction is determined by extra- or intracellular location of the infective agent. Several systems of the regulation of bacterial pathogenicity factors have been described that are relatively not numerous, conservative and respond to external signals. The relevance of a number of stressor induced proteins of bacteria to virulence associated factors is discussed.     

In vitro susceptibility to tetracycline and fluoroquinolones of Japanese isolates of Coxiella burnetii

Coxiella burnetii is the agent of the worldwide zoonosis, Q fever. The in vitro susceptibility to tetracycline and fluoroquinolones of Japanese isolates of C. burnetii was evaluated for the first time. The MICs against Japanese isolates were almost the same as the MICs against the foreign reference isolates. The results suggest that the common antibiotics therapy for Q fever used in other countries is also effective for Japanese Q fever patients.     

Pathogenicity of six isolates of Beauveria bassiana (Balsamo) Vuillemin (Deuteromycotina, Hyphomycetes) to Perillus bioculatus (F) (Hem., Pentatomidae)

Abstract:  The two-spotted stink bug, Perillus bioculatus (F) (Hem., Pentatomidae) is considered an important predator of Colorado potato beetle egg masses and small larvae. The susceptibility of P. bioculatus second instar nymphs to six isolates of Beauveria bassiana (Balsamo) Vuillemin, which are highly pathogenic to Colorado potato beetle, was tested in the laboratory. Five isolates were highly pathogenic and caused more than 77% mortality after 8 days at a concentration of 10⁶ conidia/ml. However, isolate IPP46 showed low pathogenicity and caused only 11% mortality of nymphs. The pathogenicity of isolates was independent of their host, source or country of origin. Isolate IPP46 was tested at six different concentrations. A linear relationship between the concentration of B. bassiana and the mortality of nymphs was observed. The 50% lethal concentration (LC₅₀) value was 3.4 × 10⁷ conidia/ml. The LT₅₀ decreased with increase of conidia concentration used. The present results suggest that isolate IPP46 has good potential as a biological control agent within an integrated pest management programme.     

Epidemiological investigation and physician awareness regarding the diagnosis and management of Q fever in South Korea, 2011 to 2017

BackgroundIn South Korea, the number of Q fever cases has rapidly increased since 2015. Therefore, this study aimed to characterize the epidemiological and clinical features of Q fever in South Korea between 2011 and 2017.Methods/Principal findingsWe analyzed the epidemiological investigations and reviewed the medical records from all hospitals that had reported at least one case of Q fever from 2011 to 2017. We also conducted an online survey to investigate physicians’ awareness regarding how to appropriately diagnose and manage Q fever. The nationwide incidence rate of Q fever was annually 0.07 cases per 100,000 persons. However, there has been a sharp increase in its incidence, reaching up to 0.19 cases per 100,000 persons in 2017. Q fever sporadically occurred across the country, with the highest incidences in Chungbuk (0.53 cases per 100,000 persons per year) and Chungnam (0.27 cases per 100,000 persons per year) areas. Patients with acute Q fever primarily presented with mild illnesses such as hepatitis (64.5%) and isolated febrile illness (24.0%), whereas those with chronic Q fever were likely to undergo surgery (41.2%) and had a high mortality rate (23.5%). Follow-up for 6 months after acute Q fever was performed by 24.0% of the physician respondents, and only 22.3% of them reported that clinical and serological evaluations were required after acute Q fever diagnosis.ConclusionsQ fever is becoming an endemic disease in the midwestern area of South Korea. Given the clinical severity and mortality of chronic Q fever, physicians should be made aware of appropriate diagnosis and management strategies for Q fever.     

The ligands of Numb proteins X1 and X2 are specific markers for chronic Q fever

Q fever is a disease caused by Coxiella burnetii, an obligate intracellular bacterium. Acute Q fever is spontaneously resolutive and is characterized by an efficient immune response. In contrast, chronic Q fever is characterized by dysregulated immune response, as demonstrated by the failure of C. burnetii to induce lymphoproliferation and the lack of granulomas. Recently, it has been demonstrated that when co-expressed in heterologous mammalian cell lines, the ligands of Numb proteins X1 and X2 (LNX1 and LNX2) regulate the level of the T-cell co-receptor CD8, which plays an essential role in T-cell-mediated immune response. We decided to investigate the expression of LNX1 and LNX2 genes in patients with acute or chronic Q fever. Interestingly, we found a high level of LNX1 and LNX2 mRNAs in endocarditis, the principal manifestation of chronic Q fever, but not in acute Q fever. Our data suggest that LNXs may be used as complementary biomarkers to follow the prognosis of chronic Q fever.     

Insights from the Genome Sequence of a Salmonella enterica Serovar Typhi Strain Associated with a Sporadic Case of Typhoid Fever in Malaysia

Salmonella enterica serovar Typhi is the causative agent of typhoid fever, which causes nearly 21.7 million illnesses and 217,000 deaths globally. Herein, we describe the whole-genome sequence of the Salmonella Typhi strain ST0208, isolated from a sporadic case of typhoid fever in Kuala Lumpur, Malaysia. The whole-genome sequence and comparative genomics allow an in-depth understanding of the genetic diversity, and its link to pathogenicity and evolutionary dynamics, of this highly clonal pathogen that is endemic to Malaysia.     

High endemicity of Q fever in French Guiana: A cross sectional study (2007–2017)

Q fever (QF) is a zoonosis caused by Coxiella burnetii (Cb). French Guiana (FG) had a high incidence but no data have been published since 2006. The objective of this study was to update the incidence and epidemiological data on QF in FG. A retrospective study of all FG Q fever serodiagnosis between 2007 and 2017 was carried out. Among the 695 patients included, the M/F sex-ratio was 2.0 and the median age of 45.3 years (IQR 33.7–56.3). The annual QF incidence rate was 27.4 cases (95%CI: 7.1–47.7) per 100,000 inhabitants ranging from 5.2 in 2007 to 40.4 in 2010. Risk factors associated with Q fever compared to general population were male gender, being born in mainland France, an age between 30 to 59 years-old and a residence in Cayenne and surroundings. The incidence of QF in FG remains high and stable and the highest in the world.     

Protein array of Coxiella burnetii probed with Q fever sera

Coxiella burnetii is the etiological agent of Q fever.To identify its major seroreactive proteins,a subgenomic protein array was developed.A total of 101 assumed virulence-associated recombinant proteins of C.burnetii were probed with sera from mice experimentally infected with C.burnetii and sera from Q fever patients.Sixteen proteins were recognized as major seroreactive antigens by the mouse sera.Seven of these 16 proteins reacted positively with at least 45% of Q fever patient sera.Notably,HspB had the highest fluorescence intensity value and positive frequency of all the proteins on the array when probed with both Q fever patient sera and mouse sera.These results suggest that these seven major seroreactive proteins,particularly HspB,are potential serodiagnostic and subunit vaccine antigens of Q fever.     

Frp1 is a Fusarium oxysporum F-box protein required for pathogenicity on tomato

Fusarium oxysporum f. sp. lycopersici is the causal agent of tomato wilt disease. In order to identify genes involved in its pathogenicity, we performed insertional mutagenesis. Mutant N40 had lost its pathogenicity completely, when tested in bioassays with tomato seedlings. Molecular characterization of mutant N40 revealed that the plasmid insertion had occurred in a gene that codes for a 60.2 kDa protein containing an F-box motif. The gene was therefore designated as FRP1 (F-box protein required for pathogenicity). Targeted FRP1 disruptants had lost their pathogenicity completely, and became fully virulent again upon re-introduction of the FRP1 gene. This confirmed that the FRP1 gene is required for pathogenesis. In a yeast two-hybrid assay Frp1 interacts with Skp1, suggesting involvement of an SCF ubiquitin ligase complex in pathogenicity. FRP1 is constitutively expressed during infection and under different culture conditions. Although growth, spore formation and germination on artificial media were not impaired, confocal laser scanning microscopy of a GFP-marked mutant N40 and a GFP-marked targeted FRP1 disruptant revealed that they were unable to colonize the roots.     

Profiling the humoral immune response of acute and chronic Q fever by protein microarray

Antigen profiling using comprehensive protein microarrays is a powerful tool for characterizing the humoral immune response to infectious pathogens. Coxiella burnetii is a CDC category B bioterrorist infectious agent with worldwide distribution. In order to assess the antibody repertoire of acute and chronic Q fever patients we have constructed a protein microarray containing 93% of the proteome of Coxiella burnetii, the causative agent of Q fever. Here we report the profile of the IgG and IgM seroreactivity in 25 acute Q fever patients in longitudinal samples. We found that both early and late time points of infection have a very consistent repertoire of IgM and IgG response, with a limited number of proteins undergoing increasing or decreasing seroreactivity. We also probed a large collection of acute and chronic Q fever patient samples and identified serological markers that can differentiate between the two disease states. In this comparative analysis we confirmed the identity of numerous IgG biomarkers of acute infection, identified novel IgG biomarkers for acute and chronic infections, and profiled for the first time the IgM antibody repertoire for both acute and chronic Q fever. Using these results we were able to devise a test that can distinguish acute from chronic Q fever. These results also provide a unique perspective on isotype switch and demonstrate the utility of protein microarrays for simultaneously examining the dynamic humoral immune response against thousands of proteins from a large number of patients. The results presented here identify novel seroreactive antigens for the development of recombinant protein-based diagnostics and subunit vaccines, and provide insight into the development of the antibody response.     

HA基因322位和329位氨基酸对H5N1亚型禽流感病毒毒力的影响

A/mallard/Huadong/S/2005(S,IVPI=2.65)和A/mallard/Huadong/Y/2003(Y,IVPI=O),是对麻鸭具有不同致病力的病毒.两病毒的HA裂解位点区有2个氨基酸差异,S病毒在HA裂解位点区322是Leu(L322),329位缺失(-329),而Y病毒322位是Gin(Q 322),329位是Lys(K329).根据这两个位点的差异,利用反向遗传系统,以S和Y病毒各自为骨架,拯救HA基因突变病毒,检测获救的突变病毒对麻鸭的毒力.可以得知,以S病毒为骨架,将S病毒HA基因322位Leu替换为Gln和(或)在329位添加Lys,以及用Y病毒的HA(Q322L,K329-)替换S病毒HA,获救的重组病毒对麻鸭亦完全无致病力;但以Y病毒为骨架,将Y病毒HA基因322位Gln替换为Leu和(或)在329位缺失Lys后,Y重组病毒对麻鸭的毒力上升.结果提示,S和Y病毒HA基因裂解位点区322和329氨基酸残基突变或缺失均影响病毒对麻鸭的致病力,且HA基因与其它基因的匹配性显著影响病毒对麻鸭的致病力.     

The dual function in virulence and host range restriction of a gene isolated from the pPATH (Ehg) plasmid of Erwinia herbicola pv. gypsophilae

The host range of the gall-forming bacterium Erwinia herbicola pv. gypsophilae (Ehg) is restricted to gypsophila whereas Erwinia herbicola pv. betae (Ehb) attacks beet as well as gypsophila. Both pathovars contain an indigenous plasmid (pPATH(Ehg or pPATH(Ehb)) that harbors pathogenicity genes, including the hrp gene cluster. A cosmid library of Ehg824-1 plasmid DNA was mobilized into Ehb4188 and the transconjugants were screened for pathogenicity on beet. One Ehb transconjugant harboring the cosmid pLA173 of pPATHEb induced a hypersensitive-like response and abolished pathogenicity on beet. Transposon mutagenesis of an open reading frame (ORF) located on this cosmid eliminated its affect on pathogenicity. Marker exchange of this mutation into Ehg824-1 caused a substantial reduction in gall size on gypsophila and caused Ehg824-1 to extend its host range and incite galls on beet. The ORF (1.5 kb) was designated as pthG (pathogenicity gene on gypsophila). DNA sequence analysis of pthG revealed no significant homology to known genes in the data bank. Only remnants of the pthG sequences were identified on the pPATH of Ehb4188. The deduced protein lacked an N-terminal signal peptide but contained a short trans-membrane helix in its C terminus. The gene product, as determined by expression in Escherichia coli and Western blots (immunoblots), was a 56-kDa protein.     

从功能基因到生物学性状:大丽轮枝菌致病性形成的分子基础

大丽轮枝菌为典型的土传维管束病原真菌,针对其致病相关基因的挖掘与功能解析一直是植物病理学研究的热点.大丽轮枝菌具有定殖维管束、"毒素"致萎、形成微菌核、种群分化多元化等特征,这些性状最终支撑或决定了病原对寄主的致病性基础.从进化角度来说,功能基因决定生物学性状.截至目前,在大丽轮枝菌中已鉴定出上百个功能基因;但针对其与...