Proteomic Adaptation of Streptococcus pneumoniae to the Human Antimicrobial Peptide LL-37

Secreted antimicrobial peptides (AMPs) are an important part of the human innate immune system and prevent local and systemic infections by inhibiting bacterial growth in a concentration-dependent manner. In the respiratory tract, the cationic peptide LL-37 is one of the most abundant AMPs and capable of building pore complexes in usually negatively charged bacterial membranes, leading to the destruction of bacteria. However, the adaptation mechanisms of several pathogens to LL-37 are already described and are known to weaken the antimicrobial effect of the AMP, for instance, by repulsion, export or degradation of the peptide. This study examines proteome-wide changes in Streptococcus pneumoniae D39, the leading cause of bacterial pneumonia, in response to physiological concentrations of LL-37 by high-resolution mass spectrometry. Our data indicate that pneumococci may use some of the known adaptation mechanisms to reduce the effect of LL-37 on their physiology, too. Additionally, several proteins seem to be involved in resistance to AMPs which have not been related to this process before, such as the teichoic acid flippase TacF (SPD_1128). Understanding colonization- and infection-relevant adaptations of the pneumococcus to AMPs, especially LL-37, could finally uncover new drug targets to weaken the burden of this widespread pathogen

Investigating Lactococcus lactis MG1363 response to phage p2 infection at the proteome level

Phages are viruses that specifically infect and eventually kill their bacterial hosts. Bacterial fermentation and biotechnology industries see them as enemies, however, they are also investigated as antibacterial agents for the treatment or prevention of bacterial infections in various sectors. They also play key ecological roles in all ecosystems. Despite decades of research some aspects of phage biology are still poorly understood. In this study, we used label-free quantitative proteomics to reveal the proteotypes of Lactococcus lactis MG1363 during infection by the virulent phage p2, a model for studying the biology of phages infecting Gram-positive bacteria. Our approach resulted in the high-confidence detection and quantification of 59% of the theoretical bacterial proteome, including 226 bacterial proteins detected only during phage infection and 6 proteins unique to uninfected bacteria. We also identified many bacterial proteins of differing abundance during the infection. Using this high-throughput proteomic datasets, we selected specific bacterial genes for inactivation using CRISPR-Cas9 to investigate their involvement in phage replication. One knockout mutant lacking gene llmg_0219 showed resistance to phage p2 because of a deficiency in phage adsorption. Furthermore, we detected and quantified 78% of the theoretical phage proteome and identified many proteins of phage p2 that had not been previously detected. Among others, we uncovered a conserved small phage protein (pORFN1) coded by an unannotated gene. We also applied a targeted approach to achieve greater sensitivity and identify undetected phage proteins that were expected to be present. This allowed us to follow the fate of pORF46, a small phage protein of low abundance. In summary, this work offers a unique view of the virulent phages' takeover of bacterial cells and provides novel information on phage-host interactions

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

Redirected Stress Responses in a Genome-Minimized 'midiBacillus' Strain with Enhanced Capacity for Protein Secretion

Genome engineering offers the possibility to create completely novel cell factories with enhanced properties for biotechnological applications. In recent years, genome minimization was extensively explored in the Gram-positive bacterial cell factory Bacillus subtilis, where up to 42% of the genome encoding dispensable functions was removed. Such studies showed that some strains with minimized genomes gained beneficial features, especially for secretory protein production. However, strains with the most minimal genomes displayed growth defects. This focused our attention on strains with less extensive genomic deletions that display close-to-wild-type growth properties while retaining the acquired beneficial traits in secretory protein production. A strain of this category is B. subtilis IIG-Bs27-47-24, here referred to as midiBacillus, which lacks 30.95% of the parental genome. To date, it was unknown how the altered genomic configuration of midiBacillus impacts cell physiology in general, and protein secretion in particular. The present study bridges this knowledge gap through comparative quantitative proteome analyses with focus on protein secretion. Interestingly, the results show that the secretion stress responses of midiBacillus, as elicited by high-level expression of the immunodominant staphylococcal antigen A, are completely different from secretion stress responses that occur in the parental strain 168. We further show that midiBacillus has an increased capacity for translation and that a variety of critical Sec secretion machinery components is present at elevated levels. Altogether, our observations demonstrate that high-level protein secretion has different consequences for wild-type and genome-engineered Bacillus strains, dictated by the altered genomic and proteomic configurations. IMPORTANCE Our present study showcases a genome-minimized nonpathogenic bacterium, the so-called midiBacillus, as a chassis for the development of future industrial strains that serve in the production of high-value difficult-to-produce proteins. In particular, we explain how midiBacillus, which lacks about one-third of the original genome, effectively secretes a protein of the major human pathogen Staphylococcus aureus that cannot be produced by the parental Bacillus subtilis strain. This is important, because the secreted S. aureus protein is exemplary for a range of targets that can be implemented in future antistaphylococcal immunotherapies. Accordingly, we anticipate that midiBacillus chassis will contribute to the development of vaccines that protect both humans and livestock against diseases caused by S. aureus, a bacterial pathogen that is increasingly difficult to fight with antibiotics, because it has accumulated resistances to essentially all antibiotics that are currently in clinical practice

Proteomic Investigation Uncovers Potential Targets and Target Sites of Pneumococcal Serine-Threonine Kinase StkP and Phosphatase PhpP

Like eukaryotes, different bacterial species express one or more Ser/Thr kinases and phosphatases that operate in various signaling networks by catalyzing phosphorylation and dephosphorylation of proteins that can immediately regulate biochemical pathways by altering protein function. The human pathogen Streptococcus pneumoniae encodes a single Ser/Thr kinase-phosphatase couple known as StkP-PhpP, which has shown to be crucial in the regulation of cell wall synthesis and cell division. In this study, we applied proteomics to further understand the physiological role of pneumococcal PhpP and StkP with an emphasis on phosphorylation events on Ser and Thr residues. Therefore, the proteome of the non-encapsulated D39 strain (WT), a kinase (ΔstkP), and phosphatase mutant (ΔphpP) were compared in a mass spectrometry based label-free quantification experiment. Results show that a loss of function of PhpP causes an increased abundance of proteins in the phosphate uptake system Pst. Quantitative proteomic data demonstrated an effect of StkP and PhpP on the two-component systems ComDE, LiaRS, CiaRH, and VicRK. To obtain further information on the function, targets and target sites of PhpP and StkP we combined the advantages of phosphopeptide enrichment using titanium dioxide and spectral library based data evaluation for sensitive detection of changes in the phosphoproteome of the wild type and the mutant strains. According to the role of StkP in cell division we identified several proteins involved in cell wall synthesis and cell division that are apparently phosphorylated by StkP. Unlike StkP, the physiological function of the co-expressed PhpP is poorly understood. For the first time we were able to provide a list of previously unknown putative targets of PhpP. Under these new putative targets of PhpP are, among others, five proteins with direct involvement in cell division (DivIVA, GpsB) and peptidoglycan biosynthesis (MltG, MreC, MacP)

A Metabolic Labeling Strategy for Relative Protein Quantification in Clostridioides difficile

Clostridioides difficile (formerly Clostridium difficile) is a Gram-positive, anaerobe, spore-forming pathogen, which causes drug-induced diseases in hospitals worldwide. A detailed analysis of the proteome may provide new targets for drug development or therapeutic strategies to combat this pathogen. The application of metabolic labeling (ML) would allow for accurate quantification of significant differences in protein abundance, even in the case of very small changes. Additionally, it would be possible to perform more accurate studies of the membrane or surface proteomes, which usually require elaborated sample preparation. Such studies are therefore prone to higher standard deviations during the quantification. The implementation of ML strategies for C. difficile is complicated due to the lack in arginine and lysine auxotrophy as well as the Stickland dominated metabolism of this anaerobic pathogen. Hence, quantitative proteome analyses could only be carried out by label free or chemical labeling methods so far. In this paper, a ML approach for C. difficile is described. A cultivation procedure with 15N-labeled media for strain 630Δerm was established achieving an incorporation rate higher than 97%. In a proof-of-principle experiment, the performance of the ML approach in C. difficile was tested. The proteome data of the cytosolic subproteome of C. difficile cells grown in complex medium as well as two minimal media in the late exponential and early stationary growth phase obtained via ML were compared with two label free relative quantification approaches (NSAF and LFQ). The numbers of identified proteins were comparable within the three approaches, whereas the number of quantified proteins were between 1,110 (ML) and 1,861 (LFQ) proteins. A hierarchical clustering showed clearly separated clusters for the different conditions and a small tree height with ML approach. Furthermore, it was shown that the quantification based on ML revealed significant altered proteins with small fold changes compared to the label free approaches. The quantification based on ML was accurate, reproducible, and even more sensitive compared to label free quantification strategies

An augmented reality home-training system based on the mirror training and imagery approach

Trojan J, Diers M, Fuchs X, et al. An augmented reality home-training system based on the mirror training and imagery approach. Behavior Research Methods. 2013;46(3):634-640

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

Exoproteomic profiling uncovers critical determinants for virulence of livestock-associated and human-originated Staphylococcus aureus ST398 strains

Staphylococcus aureus: with the sequence type (ST) 398 was previously associated with livestock carriage. However, in recent years livestock-independent S. aureus ST398 has emerged, representing a potential health risk for humans especially in nosocomial settings. Judged by whole-genome sequencing analyses, the livestock- and human originated strains belong to two different S. aureus ST398 clades but, to date, it was not known to what extent these clades differ in terms of actual virulence. Therefore, the objective of this study was to profile the exoproteomes of 30 representative S. aureus ST398 strains by mass spectrometry, to assess clade-specific differences in virulence factor secretion, and to correlate the identified proteins and their relative abundance to the strains' actual virulence. Although the human-originated strains are more heterogeneous at the genome level, our observations show that they are more homogeneous in terms of virulence factor production than the livestock-associated strains. To assess differences in virulence, infection models based on larvae of the wax moth Galleria mellonella and the human HeLa cell line were applied. Correlation of the exoproteome data to larval killing and toxicity toward HeLa cells uncovered critical roles of the staphylococcal Sbi, SpA, SCIN and CHIPS proteins in virulence. These findings were validated by showing that sbi or spa mutant bacteria are attenuated in G. mellonella and that the purified SCIN and CHIPS proteins are toxic for HeLa cells. Altogether, we show that exoproteome profiling allows the identification of critical determinants for virulence of livestock-associated and human-originated S. aureus ST398 strains

The glyceraldehyde-3-phosphate dehydrogenase GapDH of Corynebacterium diphtheriae is redox-controlled by protein S-mycothiolation under oxidative stress

AbstractMycothiol (MSH) is the major low molecular weight (LMW) thiol in Actinomycetes and functions in post-translational thiol-modification by protein S-mycothiolation as emerging thiol-protection and redox-regulatory mechanism. Here, we have used shotgun-proteomics to identify 26 S-mycothiolated proteins in the pathogen Corynebacterium diphtheriae DSM43989 under hypochlorite stress that are involved in energy metabolism, amino acid and nucleotide biosynthesis, antioxidant functions and translation. The glyceraldehyde-3-phosphate dehydrogenase (GapDH) represents the most abundant S-mycothiolated protein that was modified at its active site Cys153 in vivo. Exposure of purified GapDH to H2O2 and NaOCl resulted in irreversible inactivation due to overoxidation of the active site in vitro. Treatment of GapDH with H2O2 or NaOCl in the presence of MSH resulted in S-mycothiolation and reversible GapDH inactivation in vitro which was faster compared to the overoxidation pathway. Reactivation of S-mycothiolated GapDH could be catalyzed by both, the Trx and the Mrx1 pathways in vitro, but demycothiolation by Mrx1 was faster compared to Trx. In summary, we show here that S-mycothiolation can function in redox-regulation and protection of the GapDH active site against overoxidation in C. diphtheriae which can be reversed by both, the Mrx1 and Trx pathways.</jats:p