Expression, purification and characterization of the IcmG and IcmK proteins of the type IVB secretion system from Coxiella burnetii.

Coxiella burnetii, the causative agent of Q fever, is an intracellular bacterial pathogen. Studies on Coxiella have shown that a type IVB secretion system (T4BSS) contributes to the establishment of the infection by transferring protein molecules. In this report, we focus on two core proteins of the Coxiella T4BSS, namely the IcmG/DotF protein (CBU_1626) and the IcmK/DotH protein (CBU_1628). Here we present a method for the recombinant expression of IcmG and IcmK in E. coli. IcmG was purified by Strep-Tactin affinity chromatography and size exclusion chromatography, while for the purification of IcmK an additional anion exchange chromatography step was introduced. The yields of the purified IcmG and IcmK proteins were 1.2 mg/L and 3 mg/L, respectively. The purified proteins showed predominant band on SDS-PAGE gel of 37 kDa for the IcmG and 40 kDa for the IcmK. Protein folding is confirmed by circular dichroism spectroscopy. The dynamic light scattering experiment indicated that IcmG and IcmK existed in a homogenous form. Further Blue native PAGE indicates the presences of a monomeric form for the IcmK and IcmG. Our work lays the basis for functional exploration and structural determination of IcmG and IcmK proteins of Coxiella's secretion system.

In the Search of Potential Serodiagnostic Proteins to Discriminate Between Acute and Chronic Q Fever in Humans. Some Promising Outcomes.

Coxiella burnetii is the agent that causes acute and chronic Q fever infections in humans. Although the isolates studied so far have shown that the two forms of the disease differ in virulence potential thus, implying a variance in their proteomic profile, the methods used do not deliver enough discriminatory capability and often, human infections may be mis-diagnosed. The current study adds further knowledge to the results that we have already published on the Coxiella outer membrane protein 1 (Com1). Herein we identified the proteins GroEL, Ybgf, OmpH, and UPF0422 as candidates for serodiagnostics of Q fever; following cloning, expression and purification they were further used as antigens in ELISA for the screening of patients' sera associated with chronic Q fever endocarditis, sera negative for phase I IgG, sera with at least one sample positive for phase I IgG and sera from patients who suffered from various rheumatic diseases. Blood donors were used as the controls. Sensitivity, specificity, positive predictive value, negative predictive value, and Cohen's kappa coefficient (κ) were calculated and we also performed binary logistic regression analysis to identify combinations of proteins with increased diagnostic yield. We found that proteins GroEL and Ybgf, together with Com1, play the most significant role in the correct diagnosis of chronic Q fever. Of these three proteins, it was shown that Com1 and GroEL present the highest sensitivity and specificity altogether. The results add to the existing knowledge that an antigen-based serodiagnostic test that will be able to correctly diagnose chronic Q fever may not be far from reality.

Q fever in Greece: Findings of a 13 years surveillance study.

Q fever is an endemic disease in different parts of Greece. The current study aimed to investigate the prevalence of acute Q fever disease in Greece through the operation of the national reference centre for Q fever. A total of 5397 sera were received from febrile patients under the suspicion of Q fever infection during a 13 years period (2001-20013). A questionnaire was filled in by the clinicians containing certain clinical/epidemiological/demographic information. The diagnosis was based both on IFA (IgG and IgM phase II antibodies against Coxiella burnetii) and on molecular means. A total of 685 (12.7 %) samples were initially tested positive for acute Q fever. The mean (±SD) age of patients was 55.3 years (±18.7). Out of the 489 convalescent samples, 134 (27.4 %) samples indicated a minimum of a four-fold seroconversion and were considered as laboratory confirmed cases of acute Q fever. Pneumonia was the most frequently encountered clinical symptom with presence in 6.8 % of all positive samples. Forty six (46) patients were laboratory confirmed as chronic Q fever cases. Climate seemed to influence the distribution of Q fever cases throughout the years. The findings of the current study comply with past studies carried out elsewhere that had demonstrated a clear relation of the disease with temperature, south winds, etc. This study represents the first large scale attempt to gather a long period information on Q fever infection in Greece. The findings of the current study support the fact that Q fever is an important endemic zoonotic disease in Greece and needs increased awareness by clinical physicians and health care system.

Com1 as a Promising Protein for the Differential Diagnosis of the Two Forms of Q Fever.

Coxiella burnetii is the causative agent of acute and chronic Q fever in humans. Although the isolates studied so far showed a difference in virulence potential between those causing the two forms of the disease, implying a difference in their proteomic profile, the methods used so far to diagnose the two forms of the disease do not provide sufficient discriminatory capability, and human infections may be often misdiagnosed. The aim of the current study was to identify the outer membrane Com1 (CBU_1910) as a candidate protein for serodiagnostics of Q fever. The protein was cloned, expressed, purified, and used as an antigen in ELISA. The protein was then used for the screening of sera from patients suffering from chronic Q fever endocarditis, patients whose samples were negative for phase I immunoglobulin G (IgG), patients for whom at least one sample was positive for phase I IgG, and patients suffering from any kind of rheumatoid disease. Blood donors were used as the control group. Following statistical analysis, 92.4% (122/132) of the samples tested agreed with the negative clinical diagnosis, and 72.2% (26/36) agreed with the positive clinical diagnosis. Moreover, a significant correlation to the presence of the disease (p = 0.00) was calculated. The results support the idea that a Com1 antigen-based serodiagnostic test may be useful for differential diagnosis of chronic Q fever. Further studies are required to compare more immunogenic proteins of the bacterium against samples originating from patients suffering from different forms of the disease.

Use of MALDI-TOF mass spectrometry in the battle against bacterial infectious diseases: recent achievements and future perspectives.

INTRODUCTION: Advancements in microbial identification occur increasingly faster as more laboratories explore, refine and extend the use of mass spectrometry in the field of microbiology. Areas covered: This review covers the latest knowledge found in the literature for quick identification of various classes of bacterial pathogens known to cause human infection by the use of MALDI-TOF MS technology. Except for identification of bacterial strains, more researchers try to 'battle time' in favor of the patient. These novel approaches to identify bacteria directly from clinical samples and even determine antibiotic resistance are extensively revised and discussed. Expert commentary: Mass spectrometry is the future of bacterial identification and creates a new era in modern microbiology. Its incorporation in routine practice seems to be not too far, providing a valuable alternative, especially in terms of time, to conventional techniques. If the technology further advances, quick bacterial identification and probable identification of common antibiotic resistance might guide patient decision-making regarding bacterial infectious diseases in the near future.

Proteome studies of bacterial antibiotic resistance mechanisms.

Ever since antibiotics were used to help humanity battle infectious diseases, microorganisms straight away fought back. Antibiotic resistance mechanisms indeed provide microbes with possibilities to by-pass and survive the action of antibiotic drugs. Several methods have been employed to identify these microbial resistance mechanisms in an ongoing effort to reduce the steadily increasing number of treatment failures due to multi-drug-resistant microbes. Proteomics has evolved to an important tool for this area of research. Following rapid advances in whole genome sequencing, proteomic technologies have been widely used to investigate microbial gene expression. This review highlights the contribution of proteomics in identifying microbial drug resistance mechanisms. It summarizes different proteomic studies on bacteria resistant to different antibiotic drugs. The review further includes an overview of the methodologies used, as well as lists key proteins identified, thus providing the reader not only a summary of research already done, but also directions for future research. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.

Antibody kinetics in serological indication of chronic Q fever: the Greek experience.

BACKGROUND: Q fever caused by the pathogen Coxiella burnetii may have both acute and chronic manifestations. Although paired sera are not required for the diagnosis of chronic Q fever, monitoring of antibody titers can be used to examine the course of treatment. Monitoring both phase II and phase I antibodies may be of limited diagnostic value, but it is a useful means of determining the response to treatment and possible disease relapses. METHODS: In the current survey we determined IgG and IgM of both phase I and phase II for 35 patients suffering from chronic Q fever in an attempt to draw conclusions on the kinetics of the antibodies throughout the course of the disease. RESULTS: Overall, 33 cases were included in the study. Of the 33 patients, 32 had a good outcome. CONCLUSIONS: Our findings support the general belief that a long period of serological monitoring is required for patients with chronic Q fever.

The contribution of proteomics towards deciphering the enigma of Coxiella burnetii.

Coxiella burnetii, the causative agent of Q fever, is an intracellular bacterium and a potential weapon for bioterrorism. The widespread throughout the world, zoonosis is manifested clinically as a self-limited febrile illness, as pneumonia (acute Q fever) or as a chronic illness with endocarditis being its major complication. The recent Netherlands Q fever outbreak has driven the bacterium from a relatively cryptic, underappreciated, "niche" microorganism on the sideline of bacteriology, to one of possibly great impact on public health. Advances in the study of this microorganism proceeded slowly, primarily due to the, until recently, obligatory intracellular nature of the pathogen that in its virulent phase I must be manipulated under biosafety level-3 conditions. Proteomic studies, in particular, have generated a vast amount of information concerning several aspects of the bacterium such as virulence factors, detection/diagnostic and immunogenic biomarkers, inter-/intraspecies variation, resistance to antibiotics, and secreted effector proteins with significant clinical impact. The phenomenon observed following the genomics era, that of generation and accumulation of huge amount of data that ultimately end up unexploited on several databases, begins to emerge in the proteomics field as well. This review will focus on the advances in the field of C. burnetii proteomics through MS, attempting in parallel to utilize some of the proteomics findings by suggesting future directions for the improvement of Q fever diagnosis and therapy.

Study of the whole cell lysate of two Coxiella burnetii strains using N-terminomics.

The etiological agent of Q fever is Coxiella burnetii , an obligate intracellular Gram-negative bacterium and the only bacterium known to date that survives and replicates within a vacuole of phagolysosomal characteristics. In humans, Q fever is characterized by a wide spectrum of clinical manifestations. Of note is that genetic diversity among C. burnetii strains has been reported. To further investigate C. burnetii's diversity, but now at the proteome level, we compared the proteomes of whole cell lysates from two reference strains, Nine Mile and Q212. Proteomes were isolated from each strain and subjected MS-driven combined fractional diagonal chromatography (COFRADIC), a peptide-centered proteomics technique, with a total of 322 proteins that were unambiguously identified. On the basis of their identified neo-N-terminal peptides that are highly likely generated upon in vivo processing by proteases, the most proteolytical sensitive proteins in these strains were identified, and a consensus cleavage pattern was obtained. Further, with the use of differential proteomics based on the here-identified N-terminal peptides, 44 proteins were found to be differentially expressed between the two C. burnetii strains, representing 13.6% of the here-identified C. burnetii proteome. Among these proteins, 10 proteins were found uniquely expressed in the NM strain including proteins with unknown functions as well as housekeeping enzymes, suggesting that strain-related proteins might be present among such uncharacterized proteins.

Quantitative proteome profiling of C. burnetii under tetracycline stress conditions.

The recommended antibiotic regimen against Coxiella burnetii, the etiological agent of Q fever, is based on a semi-synthetic, second-generation tetracycline, doxycycline. Here, we report on the comparison of the proteomes of a C. burnetii reference strain either cultured under control conditions or under tetracycline stress conditions. Using the MS-driven combined fractional diagonal chromatography proteomics technique, out of the 531 proteins identified, 5 and 19 proteins were found significantly up- and down-regulated respectively, under tetracycline stress. Although the predicted cellular functions of these regulated proteins did not point to known tetracycline resistance mechanisms, our data clearly reveal the plasticity of the proteome of C. burnetii to battle tetracycline stress. Finally, we raise several plausible hypotheses that could further lead to more focused experiments on studying tetracycline resistance in C. burnetii and thus reduced treatment failures of Q fever.

Serological survey of Q fever in Crete, southern Greece.

Coxiella burnetii, the causative agent of Q fever, is an obligatory intracellular bacterium with worldwide distribution. The aim of this study was to determine the prevalence of C. burnetii phase II antibodies in two different groups (high and low risk) of healthy human population and investigate the epidemiological characteristics of the infection in the island of Crete (southern Greece). Collection and testing by IFA of 493 sample sera for IgG and IgM antibodies against C. bumetii phase II antigen indicated a prevalence of IgG antibodies of 48.7%. Of the seropositive individuals, 34% also revealed IgM seropositive antibody titers. Analysis of 225 sample sera by IFA from high risk population presented a prevalence for C. burnetii of 62.2%. Our findings revealed that C. burnetii is highly endemic in Crete, indicating a high exposure of the population to the pathogen regardless of occupation or place of residence.

Unraveling persistent host cell infection with Coxiella burnetii by quantitative proteomics.

The interaction between the immune system and invading bacteria is sufficient to eradicate microorganisms for the majority of bacterial infections, but suppression of the microbicidal response leads to reactivation or chronic evolution of infections and to bacterial persistence. To identify the cellular pathways affected by bacterial persistence, we applied the MS-driven combined fractional diagonal chromatography (COFRADIC) proteomics technique for a comparative study of protein expression in the C. burnetii strains Nine Mile (NM) and its respective strain (NMper) isolated from 18 months persistently infected cell cultures. In total, three different proteome comparisons were performed with the total bacterial proteome, potentially secreted bacterial proteins, and the eukaryotic infected proteome being assessed. Our results revealed that among the 547 identified bacterial proteins, 53 had significantly altered levels of expression and indicated potential metabolic differences between the two strains. Regarding differences in the secreted proteins between both strains and different modulation of the host cell, machineries reflect at least large rearrangements of both bacterial and eukaryotic proteomes during the persistent model of infection when compared to the acute one, which emphasizes that C. burnetii orchestrates a vast number of different bacterial and eukaryotic host cell processes to persist within its host.

A proteomic approach to investigate the differential antigenic profile of two Coxiella burnetii strains.

Q fever is a widespread zoonosis caused by Coxiella burnetii, an obligate intracellular Gram-negative bacterium. Current diagnostics of Q fever is based on serological testing of patient serum. Biological distinction among C. burnetii strains has been referred at the genetic level as well as in virulence in animal models of Q fever. Disclosure of strain specific antigens might show insight into the biology and pathogenesis of this query pathogen, as well as it can provide the literature with potential serodiagnostic markers. In the present study, we sought to obtain an outer membrane enriched fraction of two C. burnetii reference strains, which originate from different sources, in order to investigate the way in which their antigenic profile is differentiated against a patient serum. We systematically analyzed the sarcosyl-insoluble fraction, enriched in outer membrane proteins, of the two C. burnetii strains using doubled SDS-PAGE combined with MS/MS analysis. In total, twenty-two outer membrane proteins were identified, representing 26% of the overall 86 identified proteins. The sarcosyl-insoluble fraction was then separated on 2DE IEF/SDS-PAGE and probed with serum from an infected patient. Different immuno-reactive proteins between the two C. burnetii strains were identified and included 2 outer membrane proteins, a hypothetical protein (CBU_0937) with unknown function and OmpH (CBU_0612), a previously identified marker for Q fever endocarditis. This approach can be used to reveal strain-specific proteins involved in pathogenesis and new serodiagnostic markers.

Identification of potentially involved proteins in levofloxacin resistance mechanisms in Coxiella burnetii.

The etiological agent of Q fever, Coxiella burnetii, is an obligate intracellular bacterium that multiplies within a phagosome-like parasitophorous vacuole. Fluoroquinolones have been used as an alternative therapy for Q fever. Resistance to fluoroquinolones can arise via several mechanisms utilized by pathogens to avoid killing. Until today, genome-based studies have shown that the main mechanism of C. burnetii to resist inhibition by fluoroquinolones is based on mutations in quinolone-resistance-determining region (QRDR). In this study, in a broader search at the protein level for C. burnetii mechanisms that confer resistance to fluoroquinolones, the proteomes of in vitro developed fluoroquinolone resistant bacteria and susceptible bacteria were compared using the MS-driven combined fractional diagonal chromatography (COFRADIC) proteomics technique. Quantitative comparison of the 381 proteins identified in both strains indicated the different expression of 15 bacterial proteins. These proteins are involved in different cellular processes indicating that the antibiotic resistance mechanism of the bacterium is a multifaceted process.

DNA gyrase and topoisomerase IV mutations in an in vitro fluoroquinolone-resistant Coxiella burnetii strain.

The etiological agent of Q fever, Coxiella burnetii, is an obligate intracellular bacterium that multiplies within a vacuole with lysosomal characteristics. Quinolones have been used as an alternative therapy for Q fever. In this study, quinolone-resistance-determining regions of the genes coding for DNA gyrase and topoisomerase IV were analyzed by DNA sequencing from an in vitro fluoroquinolone-resistant C. burnetii strain (Q212). Sequencing and aligning of DNA gyrase encoding genes (gyrA and gyrB) and topoisomerase IV genes (parC and parE) revealed one gyrA mutation leading to the amino acid substitution Asp87Gly (Escherichia coli numbering), two gyrB mutations leading to the amino acid substitutions Ser431Pro and Met518Ile, and three parC mutations leading to the amino acid substitutions Asp69Asn, Thr80Ile, and Gly104Ser. The corresponding alignment of the C. burnetii Q212 reference strain, the in vitro developed fluoroquinolone-resistant C. burnetii Q212 strain, and E. coli resulted in the identification of several other naturally occurring mutations within and outside the quinolone-resistance-determining regions of C. burnetii providing indications of possible natural resistance to fluoroquinolones. The present study adds additional potential mutations in the DNA topoisomerases that may be involved in fluoroquinolone resistance in C. burnetii due to their previous characterization in other bacterial species.

Proteomic screening for possible effector molecules secreted by the obligate intracellular pathogen Coxiella burnetii.

Coxiella burnetii is a Gram-negative, gamma-proteobacteria with nearly worldwide distribution, and it is the pathogenic agent of Q-fever in man. It is an obligate intracellular parasite that is highly adapted to reside within the eukaryotic phagolysosome. In fact, it is the only known intracellular bacterium that manages to survive and replicate within a fully formed, acidic phagolysosome. C. burnetti possesses a functional Type 4 Secretion System (T4SS), similar to the Dot/Icm system of Legionella pneumophila. Up to date there have been no reports for the effector molecules secreted by Coxiella's T4SS. These are speculated to have quite different roles than the effectors of other intracellular pathogens, since there is no need for phagosomal arrest or escape in the case of Coxiella. In this study, we have investigated the cytoplasm of Vero cells infected with C. burnetti strain Nine Mile Phase II. We have identified by mass spectrometry (ESI-MS/MS) several C. burnetti proteins that bear typical characteristics of effector molecules. Most of the identified proteins were also very alkaline, something which is supportive for a protective strategy that has evolved in this bizarre pathogen against acidic environments.

Phylogentic analysis of Anaplasma ovis strains isolated from sheep and goats using groEL and mps4 genes.

Evidence of Anaplasma spp. in goats and sheep in Cyprus has been demonstrated by previous research. Herein, further research was performed for the identification of the exact Anaplasma spp. resulting in the identification of Anaplasma ovis strains in all samples examined. We used a bioinformatics as well as a molecular approach (study of groEl and mps4 genes) in order to verify the validity of the results. All samples depicted the presence of A. ovis regardless of the host (goat or sheep).

Bacteriostatic and bactericidal activities of tigecycline against Coxiella burnetii and comparison with those of six other antibiotics.

The present article is a study of the in vitro susceptibility of eight Greek Coxiella burnetii isolates, derived from patients with acute Q fever, and two reference strains of Coxiella burnetii to tigecycline. The bacteriostatic activity of tigecycline was compared with those of six other antibiotics using a shell vial assay. The MICs of the examined antibiotics were as follows: tigecycline ranged from 0.25 to 0.5 microg/ml; doxycycline, trovafloxacin, and ofloxacin ranged from 1 to 2 microg/ml; linezolid and clarithromycin ranged from 2 to 4 microg/ml; and ciprofloxacin ranged from 4 to 8 microg/ml. Tigecycline was effective in inhibiting the infection of Vero cells by C. burnetii. No bactericidal activity was observed against C. burnetii at 4 microg/ml.