Alpha-glucans from bacterial necromass indicate an intra-population loop within the marine carbon cycle.

Phytoplankton blooms provoke bacterioplankton blooms, from which bacterial biomass (necromass) is released via increased zooplankton grazing and viral lysis. While bacterial consumption of algal biomass during blooms is well-studied, little is known about the concurrent recycling of these substantial amounts of bacterial necromass. We demonstrate that bacterial biomass, such as bacterial alpha-glucan storage polysaccharides, generated from the consumption of algal organic matter, is reused and thus itself a major bacterial carbon source in vitro and during a diatom-dominated bloom. We highlight conserved enzymes and binding proteins of dominant bloom-responder clades that are presumably involved in the recycling of bacterial alpha-glucan by members of the bacterial community. We furthermore demonstrate that the corresponding protein machineries can be specifically induced by extracted alpha-glucan-rich bacterial polysaccharide extracts. This recycling of bacterial necromass likely constitutes a large-scale intra-population energy conservation mechanism that keeps substantial amounts of carbon in a dedicated part of the microbial loop.

MassSpecPreppy-An end-to-end solution for automated protein concentration determination and flexible sample digestion for proteomics applications.

In proteomics, fast, efficient, and highly reproducible sample preparation is of utmost importance, particularly in view of fast scanning mass spectrometers enabling analyses of large sample series. To address this need, we have developed the web application MassSpecPreppy that operates on the open science OT-2 liquid handling robot from Opentrons. This platform can prepare up to 96 samples at once, performing tasks like BCA protein concentration determination, sample digestion with normalization, reduction/alkylation and peptide elution into vials or loading specified peptide amounts onto Evotips in an automated and flexible manner. The performance of the developed workflows using MassSpecPreppy was compared with standard manual sample preparation workflows. The BCA assay experiments revealed an average recovery of 101.3% (SD: ± 7.82%) for the MassSpecPreppy workflow, while the manual workflow had a recovery of 96.3% (SD: ± 9.73%). The species mix used in the evaluation experiments showed that 94.5% of protein groups for OT-2 digestion and 95% for manual digestion passed the significance thresholds with comparable peptide level coefficient of variations. These results demonstrate that MassSpecPreppy is a versatile and scalable platform for automated sample preparation, producing injection-ready samples for proteomics research.

Global Protein Profiling in Processed Immunohistochemistry Tissue Sections.

Tissue sections, which are widely used in research and diagnostic laboratories and have already been examined by immunohistochemistry (IHC), may subsequently provide a resource for proteomic studies, even though only small amount of protein is available. Therefore, we established a workflow for tandem mass spectrometry-based protein profiling of IHC specimens and characterized defined brain area sections. We investigated the CA1 region of the hippocampus dissected from brain slices of adult C57BL/6J mice. The workflow contains detailed information on sample preparation from brain slices, including removal of antibodies and cover matrices, dissection of region(s) of interest, protein extraction and digestion, mass spectrometry measurement, and data analysis. The Gene Ontology (GO) knowledge base was used for further annotation. Literature searches and Gene Ontology annotation of the detected proteins verify the applicability of this method for global protein profiling using formalin-fixed and embedded material and previously used IHC slides.

Neutrophil-derived reactive agents induce a transient SpeB negative phenotype in Streptococcus pyogenes.

BACKGROUND: Streptococcus pyogenes (group A streptococci; GAS) is the main causative pathogen of monomicrobial necrotizing soft tissue infections (NSTIs). To resist immuno-clearance, GAS adapt their genetic information and/or phenotype to the surrounding environment. Hyper-virulent streptococcal pyrogenic exotoxin B (SpeB) negative variants caused by covRS mutations are enriched during infection. A key driving force for this process is the bacterial Sda1 DNase. METHODS: Bacterial infiltration, immune cell influx, tissue necrosis and inflammation in patient´s biopsies were determined using immunohistochemistry. SpeB secretion and activity by GAS post infections or challenges with reactive agents were determined via Western blot or casein agar and proteolytic activity assays, respectively. Proteome of GAS single colonies and neutrophil secretome were profiled, using mass spectrometry. RESULTS: Here, we identify another strategy resulting in SpeB-negative variants, namely reversible abrogation of SpeB secretion triggered by neutrophil effector molecules. Analysis of NSTI patient tissue biopsies revealed that tissue inflammation, neutrophil influx, and degranulation positively correlate with increasing frequency of SpeB-negative GAS clones. Using single colony proteomics, we show that GAS isolated directly from tissue express but do not secrete SpeB. Once the tissue pressure is lifted, GAS regain SpeB secreting function. Neutrophils were identified as the main immune cells responsible for the observed phenotype. Subsequent analyses identified hydrogen peroxide and hypochlorous acid as reactive agents driving this phenotypic GAS adaptation to the tissue environment. SpeB-negative GAS show improved survival within neutrophils and induce increased degranulation. CONCLUSIONS: Our findings provide new information about GAS fitness and heterogeneity in the soft tissue milieu and provide new potential targets for therapeutic intervention in NSTIs.

Migration of Acanthamoeba through Legionella biofilms is regulated by the bacterial Lqs-LvbR network, effector proteins and the flagellum.

The environmental bacterium Legionella pneumophila causes the pneumonia Legionnaires' disease. The opportunistic pathogen forms biofilms and employs the Icm/Dot type IV secretion system (T4SS) to replicate in amoebae and macrophages. A regulatory network comprising the Legionella quorum sensing (Lqs) system and the transcription factor LvbR controls bacterial motility, virulence and biofilm architecture. Here we show by comparative proteomics that in biofilms formed by the L. pneumophila ΔlqsR or ΔlvbR regulatory mutants the abundance of proteins encoded by a genomic 'fitness island', metabolic enzymes, effector proteins and flagellar components (e.g. FlaA) varies. ∆lqsR or ∆flaA mutants form 'patchy' biofilms like the parental strain JR32, while ∆lvbR forms a 'mat-like' biofilm. Acanthamoeba castellanii amoebae migrated more slowly through biofilms of L. pneumophila lacking lqsR, lvbR, flaA, a functional Icm/Dot T4SS (∆icmT), or secreted effector proteins. Clusters of bacteria decorated amoebae in JR32, ∆lvbR or ∆icmT biofilms but not in ∆lqsR or ∆flaA biofilms. The amoeba-adherent bacteria induced promoters implicated in motility (PflaA ) or virulence (PsidC , PralF ). Taken together, the Lqs-LvbR network (quorum sensing), FlaA (motility) and the Icm/Dot T4SS (virulence) regulate migration of A. castellanii through L. pneumophila biofilms, and - apart from the T4SS - govern bacterial cluster formation on the amoebae.

Streptococcus pneumoniae and Influenza A Virus Co-Infection Induces Altered Polyubiquitination in A549 Cells.

Epithelial cells are an important line of defense within the lung. Disruption of the epithelial barrier by pathogens enables the systemic dissemination of bacteria or viruses within the host leading to severe diseases with fatal outcomes. Thus, the lung epithelium can be damaged by seasonal and pandemic influenza A viruses. Influenza A virus infection induced dysregulation of the immune system is beneficial for the dissemination of bacteria to the lower respiratory tract, causing bacterial and viral co-infection. Host cells regulate protein homeostasis and the response to different perturbances, for instance provoked by infections, by post translational modification of proteins. Aside from protein phosphorylation, ubiquitination of proteins is an essential regulatory tool in virtually every cellular process such as protein homeostasis, host immune response, cell morphology, and in clearing of cytosolic pathogens. Here, we analyzed the proteome and ubiquitinome of A549 alveolar lung epithelial cells in response to infection by either Streptococcus pneumoniae D39Δcps or influenza A virus H1N1 as well as bacterial and viral co-infection. Pneumococcal infection induced alterations in the ubiquitination of proteins involved in the organization of the actin cytoskeleton and Rho GTPases, but had minor effects on the abundance of host proteins. H1N1 infection results in an anti-viral state of A549 cells. Finally, co-infection resembled the imprints of both infecting pathogens with a minor increase in the observed alterations in protein and ubiquitination abundance.

The global proteome and ubiquitinome of bacterial and viral co-infected bronchial epithelial cells.

Viral infections facilitate bacterial trafficking to the lower respiratory tract resulting in bacterial-viral co-infections. Bacterial dissemination to the lower respiratory tract is enhanced by influenza A virus induced epithelial cell damage and dysregulation of immune responses. Epithelial cells act as a line of defense and detect pathogens by a high variety of pattern recognition receptors. The post-translational modification ubiquitin is involved in almost every cellular process. Moreover, ubiquitination contributes to the regulation of host immune responses, influenza A virus uncoating and transport within host cells. We applied proteomics with a special focus on ubiquitination to assess the impact of single bacterial and viral as well as bacterial-viral co-infections on bronchial epithelial cells. We used Tandem Ubiquitin Binding Entities to enrich polyubiquitinated proteins and assess changes in the ubiquitinome. Infecting 16HBE cells with Streptococcus pyogenes led to an increased abundance of proteins related to mitochondrial translation and energy metabolism in proteome and ubiquitinome. In contrast, influenza A virus infection mainly altered the ubiquitinome. Co-infections had no additional impact on protein abundances or affected pathways. Changes in protein abundance and enriched pathways were assigned to imprints of both infecting pathogens. SIGNIFICANCE: Viral and bacterial co-infections of the lower respiratory tract are a burden for health systems worldwide. Therefore, it is necessary to elucidate the complex interplay between the host and the infecting pathogens. Thus, we analyzed the proteome and the ubiquitinome of co-infected bronchial epithelial cells to elaborate a potential synergism of the two infecting organisms. The results presented in this work can be used as a starting point for further analyses.

An Innovative Protocol for Metaproteomic Analyses of Microbial Pathogens in Cystic Fibrosis Sputum.

Hallmarks of cystic fibrosis (CF) are increased viscosity of mucus and impaired mucociliary clearance within the airways due to mutations of the cystic fibrosis conductance regulator gene. This facilitates the colonization of the lung by microbial pathogens and the concomitant establishment of chronic infections leading to tissue damage, reduced lung function, and decreased life expectancy. Although the interplay between key CF pathogens plays a major role during disease progression, the pathophysiology of the microbial community in CF lungs remains poorly understood. Particular challenges in the analysis of the microbial population present in CF sputum is (I) the inhomogeneous, viscous, and slimy consistence of CF sputum, and (II) the high number of human proteins masking comparably low abundant microbial proteins. To address these challenges, we used 21 CF sputum samples to develop a reliable, reproducible and widely applicable protocol for sputum processing, microbial enrichment, cell disruption, protein extraction and subsequent metaproteomic analyses. As a proof of concept, we selected three sputum samples for detailed metaproteome analyses and complemented and validated metaproteome data by 16S sequencing, metabolomic as well as microscopic analyses. Applying our protocol, the number of bacterial proteins/protein groups increased from 199-425 to 392-868 in enriched samples compared to nonenriched controls. These early microbial metaproteome data suggest that the arginine deiminase pathway and multiple proteases and peptidases identified from various bacterial genera could so far be underappreciated in their contribution to the CF pathophysiology. By providing a standardized and effective protocol for sputum processing and microbial enrichment, our study represents an important basis for future studies investigating the physiology of microbial pathogens in CF in vivo - an important prerequisite for the development of novel antimicrobial therapies to combat chronic recurrent airway infection in CF.

Surviving Serum: the Escherichia coli iss Gene of Extraintestinal Pathogenic E. coli Is Required for the Synthesis of Group 4 Capsule.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains constitute a serious and emerging clinical problem, as they cause a variety of infections and are usually highly antibiotic resistant. Many ExPEC strains are capable of evading the bactericidal effects of serum and causing sepsis. One critical factor for the development of septicemia is the increased serum survival (iss) gene, which is highly correlated with complement resistance and lethality. Although it is very important, the function of the iss gene has not been elucidated so far. We have been studying the serum survival of a septicemic strain of E. coli serotype O78, which has a group 4 capsule. Here, we show that the iss gene is required for the synthesis of capsules, which protect the bacteria from the bactericidal effect of complement. Moreover, we show that the deletion of the iss gene results in significantly increased binding of the complement proteins that constitute the membrane attack complex to the bacterial surface.

Proteomic Adaptation of Clostridioides difficile to Treatment with the Antimicrobial Peptide Nisin.

Clostridioides difficile is the leading cause of antibiotic-associated diarrhea but can also result in more serious, life-threatening conditions. The incidence of C. difficile infections in hospitals is increasing, both in frequency and severity, and antibiotic-resistant C. difficile strains are advancing. Against this background antimicrobial peptides (AMPs) are an interesting alternative to classic antibiotics. Information on the effects of AMPs on C. difficile will not only enhance the knowledge for possible biomedical application but may also provide insights into mechanisms of C. difficile to adapt or counteract AMPs. This study applies state-of-the-art mass spectrometry methods to quantitatively investigate the proteomic response of C. difficile 630∆erm to sublethal concentrations of the AMP nisin allowing to follow the cellular stress adaptation in a time-resolved manner. The results do not only point at a heavy reorganization of the cellular envelope but also resulted in pronounced changes in central cellular processes such as carbohydrate metabolism. Further, the number of flagella per cell was increased during the adaptation process. The potential involvement of flagella in nisin adaptation was supported by a more resistant phenotype exhibited by a non-motile but hyper-flagellated mutant.

Changing expression patterns of TonB-dependent transporters suggest shifts in polysaccharide consumption over the course of a spring phytoplankton bloom.

Algal blooms produce large quantities of organic matter that is subsequently remineralised by bacterial heterotrophs. Polysaccharide is a primary component of algal biomass. It has been hypothesised that individual bacterial heterotrophic niches during algal blooms are in part determined by the available polysaccharide substrates present. Measurement of the expression of TonB-dependent transporters, often specific for polysaccharide uptake, might serve as a proxy for assessing bacterial polysaccharide consumption over time. To investigate this, we present here high-resolution metaproteomic and metagenomic datasets from bacterioplankton of the 2016 spring phytoplankton bloom at Helgoland island in the southern North Sea, and expression profiles of TonB-dependent transporters during the bloom, which demonstrate the importance of both the Gammaproteobacteria and the Bacteroidetes as degraders of algal polysaccharide. TonB-dependent transporters were the most highly expressed protein class, split approximately evenly between the Gammaproteobacteria and Bacteroidetes, and totalling on average 16.7% of all detected proteins during the bloom. About 93% of these were predicted to take up organic matter, and for about 12% of the TonB-dependent transporters, we predicted a specific target polysaccharide class. Most significantly, we observed a change in substrate specificities of the expressed transporters over time, which was not reflected in the corresponding metagenomic data. From this, we conclude that algal cell wall-related compounds containing fucose, mannose, and xylose were mostly utilised in later bloom stages, whereas glucose-based algal and bacterial storage molecules including laminarin, glycogen, and starch were used throughout. Quantification of transporters could therefore be key for understanding marine carbon cycling.

Optimized Proteomics Workflow for the Detection of Small Proteins.

Small open reading frame encoded proteins (SEPs) gained increasing interest during the last few years because of their broad range of important functions in both prokaryotes and eukaryotes. In bacteria, signaling, virulence, and regulation of enzyme activities have been associated with SEPs. Nonetheless, the number of SEPs detected in large-scale proteome studies is often low as classical methods are biased toward the identification of larger proteins. Here, we present a workflow that allows enhanced identification of small proteins compared to traditional protocols. For this aim, the steps of small protein enrichment, proteolytic digest, and database search were reviewed and adjusted to the special requirement of SEPs. Enrichment by the use of small-pore-sized solid-phase material increased the number of identified SEPs by a factor of 2, and utilization of alternative proteases to trypsin reduced the spectral counts for larger proteins. The application of the optimized protocol allowed the detection of 210 already annotated proteins up to 100 amino acids (aa) length, including 16 proteins below 51 aa in the Gram-positive model organism Bacillus subtilis. Moreover, 12% of all identified proteins were up to 100 aa, which is a significantly larger fraction than that reported in studies involving traditional proteomics workflows. Finally, the application of an integrated proteogenomics search database and extensive subsequent validation resulted in the confident identification of three novel, not yet annotated, SEPs, which are 21, 26, and 42 aa long.

Membrane Modulation of Super-Secreting "midiBacillus" Expressing the Major Staphylococcus aureus Antigen - A Mass-Spectrometry-Based Absolute Quantification Approach.

Bacillus subtilis has been extensively used as a microbial cell factory for industrial enzymes due to its excellent capacities for protein secretion and large-scale fermentation. This bacterium is also an attractive host for biopharmaceutical production. However, the secretion potential of this organism is not fully utilized yet, mostly due to a limited understanding of critical rearrangements in the membrane proteome upon high-level protein secretion. Recently, it was shown that bottlenecks in heterologous protein secretion can be resolved by genome minimization. Here, we present for the first time absolute membrane protein concentrations of a genome-reduced B. subtilis strain ("midiBacillus") expressing the immunodominant Staphylococcus aureus antigen A (IsaA). We quantitatively characterize the membrane proteome adaptation of midiBacillus during production stress on the level of molecules per cell for more than 400 membrane proteins, including determination of protein concentrations for ∼61% of the predicted transporters. We demonstrate that ∼30% of proteins with unknown functions display a significant increase in abundance, confirming the crucial role of membrane proteins in vital biological processes. In addition, our results show an increase of proteins dedicated to translational processes in response to IsaA induction. For the first time reported, we provide accumulation rates of a heterologous protein, demonstrating that midiBacillus secretes 2.41 molecules of IsaA per minute. Despite the successful secretion of this protein, it was found that there is still some IsaA accumulation occurring in the cytosol and membrane fraction, leading to a severe secretion stress response, and a clear adjustment of the cell's array of transporters. This quantitative dataset offers unprecedented insights into bioproduction stress responses in a synthetic microbial cell.

Ariadne's Thread in the Analytical Labyrinth of Membrane Proteins: Integration of Targeted and Shotgun Proteomics for Global Absolute Quantification of Membrane Proteins.

The field of systems biology has been rapidly developing in the past decade. However, the data produced by "omics" approaches is lagging behind the requirements of this field, especially when it comes to absolute abundances of membrane proteins. In the present study, a novel approach for large-scale absolute quantification of this challenging subset of proteins has been established and evaluated using osmotic stress management in the Gram-positive model bacterium Bacillus subtilis as proof-of-principle precedent. Selected membrane proteins were labeled using a SNAP-tag, which allowed us to visually inspect the enrichment of the membrane fraction by immunoassays. Absolute membrane protein concentrations were determined via shotgun proteomics by spiking crude membrane extracts of chromosomally SNAP-tagged and wild-type B. subtilis strains with protein standards of known concentration. Shotgun data was subsequently calibrated by targeted mass spectrometry using SNAP as an anchor protein, and an enrichment factor was calculated in order to obtain membrane protein copy numbers per square micrometer. The presented approach enabled the accurate determination of physiological changes resulting from imposed hyperosmotic stress, thereby offering a clear visualization of alterations in membrane protein arrangements and shedding light on putative membrane complexes. This straightforward and cost-effective methodology for quantitative proteome studies can be implemented by any research group with mass spectrometry expertise. Importantly, it can be applied to the full spectrum of physiologically relevant conditions, ranging from environmental stresses to the biotechnological production of small molecules and proteins, a field heavily relying on B. subtilis secretion capabilities.

Comparative proteome analysis in an Escherichia coli CyDisCo strain identifies stress responses related to protein production, oxidative stress and accumulation of misfolded protein.

BACKGROUND: The Twin-arginine translocation (Tat) pathway of Escherichia coli has great potential for the export of biopharmaceuticals to the periplasm due to its ability to transport folded proteins, and its proofreading mechanism that allows correctly folded proteins to translocate. Coupling the Tat-dependent protein secretion with the formation of disulfide bonds in the cytoplasm of E. coli CyDisCo provides a powerful platform for the production of industrially challenging proteins. In this study, we investigated the effects on the E. coli cells of exporting a folded substrate (scFv) to the periplasm using a Tat signal peptide, and the effects of expressing an export-incompetent misfolded variant. RESULTS: Cell growth is decreased when either the correctly folded or misfolded scFv is expressed with a Tat signal peptide. However, only the production of misfolded scFv leads to cell aggregation and formation of inclusion bodies. The comprehensive proteomic analysis revealed that both conditions, recombinant protein overexpression and misfolded protein accumulation, lead to downregulation of membrane transporters responsible for protein folding and insertion into the membrane while upregulating the production of chaperones and proteases involved in removing aggregates. These conditions also differentially affect the production of transcription factors and proteins involved in DNA replication. The most distinct stress response observed was the cell aggregation caused by elevated levels of antigen 43. Finally, Tat-dependent secretion causes an increase in tatA expression only after induction of protein expression, while the subsequent post-induction analysis revealed lower tatA and tatB expression levels, which correlate with lowered TatA and TatB protein abundance. CONCLUSIONS: The study identified characteristic changes occurring as a result of the production of both a folded and a misfolded protein, but also highlights an exclusive unfolded stress response. Countering and compensating for these changes may result in higher yields of pharmaceutically relevant proteins exported to the periplasm.

Far-reaching cellular consequences of tat deletion in Escherichia coli revealed by comprehensive proteome analyses.

In Escherichia coli, the Twin-arginine translocation (Tat) pathway secretes a set of folded proteins with important physiological functions to the periplasm and outer membrane. The loss of Tat secretion impairs outer membrane integrity and leads to decreased cell growth. Only recently, the Tat pathway has gained more attention due to its essential role in bacterial virulence and applications in the production of fully folded heterologous proteins. In this study, we investigated the influence of the deletion of all active Tat pathway components on the E. coli cells. The comprehensive proteomic analysis revealed activation of several stress responses and experimentally confirmed the dependence of certain proteins on the Tat system for export. We observed that a tat deletion triggers protein aggregation, membrane vesiculation, synthesis of colanic acid and biofilm formation. Furthermore, the mislocalization of Tat-dependent proteins disturbs iron and molybdenum homeostasis and impairs the cell envelope integrity. The results show that the functional Tat pathway is important for the physiological stability and that its dysfunction leads to a series of severe changes in E. coli cells.

A Secreted Bacterial Peptidylarginine Deiminase Can Neutralize Human Innate Immune Defenses.

The keystone oral pathogen Porphyromonas gingivalis is associated with severe periodontitis. Intriguingly, this bacterium is known to secrete large amounts of an enzyme that converts peptidylarginine into citrulline residues. The present study was aimed at identifying possible functions of this citrullinating enzyme, named Porphyromonas peptidylarginine deiminase (PPAD), in the periodontal environment. The results show that PPAD is detectable in the gingiva of patients with periodontitis, and that it literally neutralizes human innate immune defenses at three distinct levels, namely bacterial phagocytosis, capture in neutrophil extracellular traps (NETs), and killing by the lysozyme-derived cationic antimicrobial peptide LP9. As shown by mass spectrometry, exposure of neutrophils to PPAD-proficient bacteria reduces the levels of neutrophil proteins involved in phagocytosis and the bactericidal histone H2. Further, PPAD is shown to citrullinate the histone H3, thereby facilitating the bacterial escape from NETs. Last, PPAD is shown to citrullinate LP9, thereby restricting its antimicrobial activity. The importance of PPAD for immune evasion is corroborated in the infection model Galleria mellonella, which only possesses an innate immune system. Together, the present observations show that PPAD-catalyzed protein citrullination defuses innate immune responses in the oral cavity, and that the citrullinating enzyme of P. gingivalis represents a new type of bacterial immune evasion factor.IMPORTANCE Bacterial pathogens do not only succeed in breaking the barriers that protect humans from infection, but they also manage to evade insults from the human immune system. The importance of the present study resides in the fact that protein citrullination is shown to represent a new bacterial mechanism for immune evasion. In particular, the oral pathogen P. gingivalis employs this mechanism to defuse innate immune responses by secreting a protein-citrullinating enzyme. Of note, this finding impacts not only the global health problem of periodontitis, but it also extends to the prevalent autoimmune disease rheumatoid arthritis, which has been strongly associated with periodontitis, PPAD activity, and loss of tolerance against citrullinated proteins, such as the histone H3.

The Escherichia coli Type III Secretion System 2 Has a Global Effect on Cell Surface.

Many strains of Escherichia coli carry a 29,250-bp ETT2 pathogenicity island (PAI), which includes genes predicted to encode type III secretion system (T3SS) components. Because it is similar to the Salmonella pathogenicity island 1 (SPI-1) system, encoding a T3SS in Salmonella enterica, it was assumed that ETT2 also encodes a secretion system injecting effectors into host cells. This assumption was checked in E. coli serotype O2-associated with urinary tract infections and septicemia-which has an intact ETT2 gene cluster, in contrast to most strains in which this cluster carries deletions and mutations. A proteomic search did not reveal any putative secreted effector. Instead, the majority of the secreted proteins were identified as flagellar proteins. A deletion of the ETT2 gene cluster significantly reduced the secretion of flagellar proteins, resulting in reduced motility. There was also a significant reduction in the transcriptional level of flagellar genes, indicating that ETT2 affects the synthesis, rather than secretion, of flagellar proteins. The ETT2 deletion also resulted in additional major changes in secretion of fimbrial proteins and cell surface proteins, resulting in relative resistance to detergents and hydrophobic antibiotics (novobiocin), secretion of large amounts of outer membrane vesicles (OMVs), and altered multicellular behavior. Most important, the ETT2 deletion mutants were sensitive to serum. These major changes indicate that the ETT2 gene cluster has a global effect on cell surface and physiology, which is especially important for pathogenicity, as it contributes to the ability of the bacteria to survive serum and cause sepsis.IMPORTANCE Drug-resistant extraintestinal pathogenic E. coli (ExPEC) strains are major pathogens, especially in hospital- and community-acquired infections. They are the major cause of urinary tract infections and are often involved in septicemia with high mortality. ExPEC strains are characterized by broad-spectrum antibiotic resistance, and development of a vaccine is not trivial because the ExPEC strains include a large number of serotypes. It is therefore important to understand the virulence factors that are involved in pathogenicity of ExPEC and identify new targets for development of antibacterial drugs or vaccines. Such a target could be ETT2, a unique type III secretion system present (complete or in parts) in many ExPEC strains. Here, we show that this system has a major effect on the bacterial surface-it affects sensitivity to drugs, motility, and secretion of extracellular proteins and outer membrane vesicles. Most importantly, this system is important for serum resistance, a prerequisite for septicemia.

Proteomic response of Streptococcus pneumoniae to iron limitation.

Iron is an essential trace element and involved in various key metabolic pathways in bacterial lifestyle. Within the human host, iron is extremely limited. Hence, the ability of bacteria to acquire iron from the environment is critical for a successful infection. Streptococcus pneumoniae (the pneumococcus) is a human pathobiont colonizing symptomless the human respiratory tract, but can also cause various local and invasive infections. To survive and proliferate pneumococci have therefore to adapt their metabolism and virulence factor repertoire to different host compartments. In this study, the response of S. pneumoniae to iron limitation as infection-relevant condition was investigated on the proteome level. The iron limitation was induced by application of the iron chelator 2,2'-bipyridine (BIP) in two different media mimicking different physiological traits. Under these conditions, the influence of the initial iron concentration on pneumococcal protein expression in response to limited iron availability was analyzed. Interestingly, one major difference between these two iron limitation experiments is the regulation of proteins involved in pneumococcal pathogenesis. In iron-poor medium several proteins of this group were downregulated whereas these proteins are upregulated in iron-rich medium. However, iron limitation in both environments led to a strong upregulation of the iron uptake protein PiuA and the significant downregulation of the non-heme iron-containing ferritin Dpr. Based on the results, it is shown that the pneumococcal proteome response to iron limitation is strongly dependent on the initial iron concentration in the medium or the environment.

Proteomics of septicemic Escherichia coli.

Virulent strains of Escherichia coli have become a major cause of infections, especially in hospitals and institutions, and result in high morbidity and mortality, due to the widespread antibiotic resistance. The infections usually start as complications of urinary tract infections or invasive medical procedures. Septicemic bacteria have to go through the blood stream, where they are exposed to a variety of stress conditions. The most difficult of these is the presence of the immune complement, which is strongly bactericidal. However, recently it has become clear that the nutritional immunity (metabolic stress) of serum is just as important. Thus, as shown by proteomic analyses, septicemic E. coli can cope with this latter stress condition by globally modifying the expression of a variety of metabolic genes. These include genes involved in amino acid metabolism and in metal homeostasis, whose robust regulation of expression appears to be critical for surviving the metabolic immunity of serum. Recognition of the nutritional immunity and the molecular mechanisms that enable septicemic bacteria to overcome it are the focus of this paper.