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In this study, we have performed a systematic analysis of Pseudomonas sp. strain phDV1 membrane protein complexes by growing the strain in lysogeny broth medium, and medium containing glucose or phenol as sole carbon sources. In order to study the membrane complexome, we developed an approach for the extraction and the analysis of the membrane protein complexes in native conditions. Our strategy involves (a) enrichment of the membrane proteome from Pseudomonas sp. strain phDV1 by two washing steps; (b) solubilization using n-dodecyl-β-maltoside; (c) a combination of BN-PAGE with Tricine-SDS-PAGE; and (d) protein identification of tryptic peptides by mass spectrometry.

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 bacterial membrane proteome is a field of growing interest in the research of nutrient transport and processing. Pseudomonas sp. strain phDV1, a Gram-negative bacterium selected for its ability to degrade aromatic compounds, was monitored under different growth substrate conditions, using lysogeny broth medium (LB), glucose, and phenol as sole carbon source. The aim of this study was to characterize the membrane subproteomes of the Pseudomonas strain by proteomic means to assess the protein composition of this subcellular compartments, which appears fundamental for the biodegradation of aromatic compounds. A total number of 129 different proteins have been identified by MALDI-TOF/TOF, 19 of which are membrane proteins that belong to the inner membrane and 10 that belong to the outer membrane. Two membrane proteins were only expressed in the presence of the aromatic substrate. We identified a membrane protein involved in aromatic hydrocarbon degradation as well as a probable porin which may, in fact, function as an aromatic compound-specific porin. Although the presence of different transporters have been reported for different aromatic compounds such as toluene and benzoic acid, to our knowledge, these are the first phenol-inducible membrane transporters identified.

Background During the Hajj season, respiratory symptoms are very common among pilgrims. Here, we investigated the viable bacterial population in air samples collected around the slaughterhouses used during the Hajj. Methods and results We collected air samples on three days from four different sites: slaughterhouses at Al-Kakia, Al-Meaisim and Al-Sharaia, and from a waste disposal area designated for the remnants of slaughter. Samples were cultured on blood agar plates for 48 h, and bacterial isolates were identified using MALDI-TOF MS. A dendrogram using the spectra of the unidentified bacterial species was constructed, and PCR amplification and sequencing of the 16S rRNA gene was performed for one isolate per cluster. In total, 2500 colonies appeared on the nutrient agar plates, and 244 were purified for further analysis. Good identification was obtained for 202 (83%) isolates by MALDI-TOF MS. The most common genera were Bacillus (n = 94, 45%) and Staphyloccocus (n = 55, 26%). Poor identification was obtained for 42 (17%) isolates, and their spectra clustering revealed that these isolates belonged to 10 species. Four of these were considered to be new species. Conclusions During the Hajj, the air was contaminated by many environmental bacterial agents, and MALDI-TOF MS was successfully adapted for their rapid identification.

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.

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.

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.

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.

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.