Comparative Omics Analysis of Historic and Recent Isolates of Bordetella pertussis and Effects of Genome Rearrangements on Evolution.

Despite high vaccination coverage, pertussis is increasing in many industrialized countries, including the Czech Republic. To better understand Bordetella pertussis resurgence, we analyzed historic strains and recent clinical isolates by using a comparative omics approach. Whole-genome sequencing showed that historic and recent isolates of B. pertussis have substantial variation in genome organization and form separate phylogenetic clusters. Subsequent RNA sequence analysis and liquid chromatography with mass tandem spectrometry analyses showed that these variations translated into discretely separated transcriptomic and proteomic profiles. When compared with historic strains, recent isolates showed increased expression of flagellar genes and genes involved in lipopolysaccharide biosynthesis and decreased expression of polysaccharide capsule genes. Compared with reference strain Tohama I, all strains had increased expression and production of the type III secretion system apparatus. We detected the potential link between observed effects and insertion sequence element-induced changes in gene context only for a few genes.

Omics Analysis of Blood-Responsive Regulon in Bordetella pertussis Identifies a Novel Essential T3SS Substrate.

Bacterial pathogens sense specific cues associated with different host niches and integrate these signals to appropriately adjust the global gene expression. Bordetella pertussis is a Gram-negative, strictly human pathogen of the respiratory tract and the etiological agent of whooping cough (pertussis). Though B. pertussis does not cause invasive infections, previous results indicated that this reemerging pathogen responds to blood exposure. Here, omics RNA-seq and LC-MS/MS techniques were applied to determine the blood-responsive regulon of B. pertussis. These analyses revealed that direct contact with blood rewired global gene expression profiles in B. pertussis as the expression of almost 20% of all genes was significantly modulated. However, upon loss of contact with blood, the majority of blood-specific effects vanished, with the exception of several genes encoding the T3SS-secreted substrates. For the first time, the T3SS regulator BtrA was identified in culture supernatants of B. pertussis. Furthermore, proteomic analysis identified BP2259 protein as a novel secreted T3SS substrate, which is required for T3SS functionality. Collectively, presented data indicate that contact with blood represents an important cue for B. pertussis cells.

Transcriptional profiling of human macrophages during infection with Bordetella pertussis.

Bordetella pertussis, a strictly human re-emerging pathogen and the causative agent of whooping cough, exploits a broad variety of virulence factors to establish efficient infection. Here, we used RNA sequencing to analyse the changes in gene expression profiles of human THP-1 macrophages resulting from B. pertussis infection. In parallel, we attempted to determine the changes in intracellular B. pertussis-specific transcriptomic profiles resulting from interaction with macrophages. Our analysis revealed that global gene expression profiles in THP-1 macrophages are extensively rewired 6 h post-infection. Among the highly expressed genes, we identified those encoding cytokines, chemokines, and transcription regulators involved in the induction of the M1 and M2 macrophage polarization programmes. Notably, several host genes involved in the control of apoptosis and inflammation which are known to be hijacked by intracellular bacterial pathogens were overexpressed upon infection. Furthermore, in silico analyses identified large temporal changes in expression of specific gene subsets involved in signalling and metabolic pathways. Despite limited numbers of the bacterial reads, we observed reduced expression of majority of virulence factors and upregulation of several transcriptional regulators during infection suggesting that intracellular B. pertussis cells switch from virulent to avirulent phase and actively adapt to intracellular environment, respectively.

Comparative Integrated Omics Analysis of the Hfq Regulon in Bordetella pertussis.

Bordetella pertussis is a Gram-negative strictly human pathogen of the respiratory tract and the etiological agent of whooping cough (pertussis). Previously, we have shown that RNA chaperone Hfq is required for virulence of B. pertussis. Furthermore, microarray analysis revealed that a large number of genes are affected by the lack of Hfq. This study represents the first attempt to characterize the Hfq regulon in bacterial pathogen using an integrative omics approach. Gene expression profiles were analyzed by RNA-seq and protein amounts in cell-associated and cell-free fractions were determined by LC-MS/MS technique. Comparative analysis of transcriptomic and proteomic data revealed solid correlation (r2 = 0.4) considering the role of Hfq in post-transcriptional control of gene expression. Importantly, our study confirms and further enlightens the role of Hfq in pathogenicity of B. pertussis as it shows that Δhfq strain displays strongly impaired secretion of substrates of Type III secretion system (T3SS) and substantially reduced resistance to serum killing. On the other hand, significantly increased production of proteins implicated in transport of important metabolites and essential nutrients observed in the mutant seems to compensate for the physiological defect introduced by the deletion of the hfq gene.

Mutual interactions of Pleurotus ostreatus with bacteria of activated sludge in solid-bed bioreactors.

White rot fungi are well known for their ability to degrade xenobiotics in pure cultures but few studies focus on their performance under bacterial stress in real wastewaters. This study investigated mutual interactions in co-cultures of Pleurotus ostreatus and activated sludge microbes in batch reactors and different culture media. Under the bacterial stress an increase in the dye decolorization efficiency (95 vs. 77.1 %) and a 2-fold elevated laccase activity (156.7 vs. 78.4 Ul(-1)) were observed in fungal-bacterial cultures compared to pure P. ostreatus despite a limited growth of bacteria in mixed cultures. According to 16S-rDNA analyses, P. ostreatus was able to alter the structure of bacterial communities. In malt extract-glucose medium the fungus inhibited growth of planktonic bacteria and prevented shifts in bacterial utilization of potential C-sources. A model bacterium, Rhodococcus erythropolis responded to fungal metabolites by down regulation of uridylate kinase and acetyl-CoA synthetase.

Morphological and proteomic analysis of early stage air-liquid interface biofilm formation in Mycobacterium smegmatis.

We studied the early stages of pellicle formation by Mycobacterium smegmatis on the surface of a liquid medium [air-liquid interface (A-L)]. Using optical and scanning electron microscopy, we showed the formation of a compact biofilm pellicle from micro-colonies over a period of 8-30 h. The cells in the pellicle changed size and cell division pattern during this period. Based on our findings, we created a model of M. smegmatis A-L early pellicle formation showing the coordinate growth of cells in the micro-colonies and in the homogeneous film between them, where the accessibility to oxygen and nutrients is different. A proteomic approach utilizing high-resolution two-dimensional gel electrophoresis, in combination with mass spectrometry-based protein identification, was used to analyse the protein expression profiles of the different morphological stages of the pellicle. The proteins identified formed four expression groups; the most interesting of these groups contained the proteins with highest expression in the biofilm development phase, when the floating micro-colonies containing long and more robust cells associate into flocs and start to form a compact pellicle. The majority of these proteins, including GroEL1, are involved in cell wall synthesis or modification, mostly through the involvement of mycolic acid biosynthesis, and their expression maxima correlated with the changes in cell size and the rigidity of the bacterial cell wall observed by scanning electron microscopy.

Stem-loop induced ribosome queuing in the uORF2/ATF4 overlap fine-tunes stress-induced human ATF4 translational control.

ATF4 is a master transcriptional regulator of the integrated stress response leading cells towards adaptation or death. ATF4's induction under stress was thought to be mostly due to delayed translation reinitiation, where the reinitiation-permissive uORF1 plays a key role. Accumulating evidence challenging this mechanism as the sole source of ATF4 translation control prompted us to investigate additional regulatory routes. We identified a highly conserved stem-loop in the uORF2/ATF4 overlap, immediately preceded by a near-cognate CUG, which introduces another layer of regulation in the form of ribosome queuing. These elements explain how the inhibitory uORF2 can be translated under stress, confirming prior observations, but contradicting the original regulatory model. We also identified two highly conserved, potentially modified adenines performing antagonistic roles. Finally, we demonstrate that the canonical ATF4 translation start site is substantially leaky-scanned. Thus, ATF4's translational control is more complex than originally described underpinning its key role in diverse biological processes.

Stem-loop-induced ribosome queuing in the uORF2/ATF4 overlap fine-tunes stress-induced human ATF4 translational control.

Activating transcription factor 4 (ATF4) is a master transcriptional regulator of the integrated stress response, leading cells toward adaptation or death. ATF4's induction under stress was thought to be due to delayed translation reinitiation, where the reinitiation-permissive upstream open reading frame 1 (uORF1) plays a key role. Accumulating evidence challenging this mechanism as the sole source of ATF4 translation control prompted us to investigate additional regulatory routes. We identified a highly conserved stem-loop in the uORF2/ATF4 overlap, immediately preceded by a near-cognate CUG, which introduces another layer of regulation in the form of ribosome queuing. These elements explain how the inhibitory uORF2 can be translated under stress, confirming prior observations but contradicting the original regulatory model. We also identified two highly conserved, potentially modified adenines performing antagonistic roles. Finally, we demonstrated that the canonical ATF4 translation start site is substantially leaky scanned. Thus, ATF4's translational control is more complex than originally described, underpinning its key role in diverse biological processes.

Fungal microsomes in a biotransformation perspective: protein nature of membrane-associated reactions.

Microsomal fraction of fungal cells grabs the attention of many researchers for it contains enzymes that play a role in biotechnologically relevant processes. Microsomal enzymes, namely, CYP450s, were shown to metabolize a wide range of xenobiotic compounds, including PAHs, PCBs, dioxins, and endocrine disruptors, and take part in other fungal biotransformation reactions. However, little is known about the nature and regulation of these membrane-associated reactions. Advanced proteomic and post-genomic techniques make it possible to identify larger numbers of microsomal proteins and thus add to a deeper study of fungal intracellular processes. In this work, proteins that were identified through a shotgun proteomic approach in fungal microsomes under various culture conditions are reviewed. However, further research is still needed to fully understand the role of microsomes in fungal biodegradation and biotransformation reactions.

T3SS chaperone of the CesT family is required for secretion of the anti-sigma factor BtrA in Bordetella pertussis.

Bordetella pertussis is a Gram-negative, strictly human re-emerging respiratory pathogen and the causative agent of whooping cough. Similar to other Gram-negative pathogens, B. pertussis produces the type III secretion system, but its role in the pathogenesis of B. pertussis is enigmatic and yet to be elucidated. Here, we combined RNA-seq, LC-MS/MS, and co-immunoprecipitation techniques to identify and characterize the novel CesT family T3SS chaperone BP2265. We show that this chaperone specifically interacts with the secreted T3SS regulator BtrA and represents the first non-flagellar chaperone required for the secretion of an anti-sigma factor. In its absence, secretion but not production of BtrA and most T3SS substrates is severely impaired. It appears that the role of BtrA in regulating T3SS extends beyond its activity as an antagonist of the sigma factor BtrS. Predictions made by artificial intelligence system AlphaFold support the chaperone function of BP2265 towards BtrA and outline the structural basis for the interaction of BtrA with its target BtrS. We propose to rename BP2265 to BtcB for the Bordetella type III chaperone of BtrA.In addition, the absence of the BtcB chaperone results in increased expression of numerous flagellar genes and several virulence genes. While increased production of flagellar proteins and intimin BipA translated into increased biofilm formation by the mutant, enhanced production of virulence factors resulted in increased cytotoxicity towards human macrophages. We hypothesize that these phenotypic traits result indirectly from impaired secretion of BtrA and altered activity of the BtrA/BtrS regulatory node.

Avirulent phenotype promotes Bordetella pertussis adaptation to the intramacrophage environment.

Bordetella pertussis, the causative agent of whooping cough, is an extracellular, strictly human pathogen. However, it has been shown that B. pertussis cells can escape phagocytic killing and survive in macrophages upon internalization. Our time-resolved RNA-seq data suggest that B. pertussis efficiently adapts to the intramacrophage environment and responds to host bactericidal activities. We show that this adaptive response is multifaceted and, surprisingly, related to the BvgAS two-component system, a master regulator of virulence. Our results show that the expression of this regulatory circuit is downregulated upon internalization. Moreover, we demonstrate that the switch to the avirulent Bvg- phase augments a very complex process based on the adjustment of central and energy metabolism, cell wall reinforcement, maintenance of appropriate redox and metal homeostasis, and repair of damaged macromolecules. Nevertheless, not all observed effects could be simply attributed to the transition to Bvg- phase, suggesting that additional regulators are involved in the adaptation to the intramacrophage environment. Interestingly, a large number of genes required for the metabolism of sulphur were strongly modulated within macrophages. In particular, the mutant lacking two genes encoding cysteine dioxygenases displayed strongly attenuated cytotoxicity toward THP-1 cells. Collectively, our results suggest that intracellular B. pertussis cells have adopted the Bvg- mode to acclimate to the intramacrophage environment and respond to antimicrobial activities elicited by THP-1 cells. Therefore, we hypothesize that the avirulent phase represents an authentic phenotype of internalized B. pertussis cells.

Long-term adaptation of Bacillus subtilis 168 to extreme pH affects chemical and physical properties of the cellular membrane.

We characterized physical and chemical properties of cell-membrane fragments from Bacillus subtilis 168 (trpC2) grown at pH 5.0, 7.0 and 8.5. Effects of long-term bacterial adaptation reflected in growth rates and in changes of the membrane lipid composition were correlated with lipid order and dynamics using time-resolved fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. We demonstrate that the pH adaptation results in a modification of a fatty acid content of cellular membranes that significantly influences both the lipid-chain order and dynamics. For cultivation at acidic conditions, the lipid order increases and membrane dynamics decreases compared to pH 7.0. This results in rigid and ordered membranes. Cultivation at pH 8.5 causes slight membrane disordering. Instant pH changes induce qualitatively similar but smaller effects. Proton flux measurements performed on intact cells adapted to both pH 5.0 and 8.5 revealed lower cell-membrane permeability compared to bacteria cultivated at pH optimum. Our results indicate that both acidic and alkalic pH stress represent a permanent challenge for B. subtilis to keep a functional membrane state. The documented adaptation-induced adjustments of membrane properties could be an important part of mechanisms maintaining an optimal intracellular pH at a wide range of extracellular proton concentrations.

PynA is a pyrimidine 5'-nucleotidase that functions as an antimutator protein in Streptococcus pneumoniae.

Streptococcus pneumoniae is a Gram-positive bacterium that is a major agent of community-acquired bacterial pneumonia, meningitis and sepsis. Although the mismatch repair function of S. pneumoniae has been assigned to the hexA-hexB gene products, an enzyme capable of the direct elimination of noncanonical nucleotides from the cytoplasm has not been described for this bacterium. Our results show that Spr1057, a protein with previously unknown function, is involved in the inactivation of mutagenic pyrimidine nucleotides and was accordingly designated PynA (pyrimidine nucleotidase A). Biochemical assays confirmed the phosphatase activity of the recombinant enzyme and revealed its metal ion dependence for optimal enzyme activity. We demonstrated that PynA forms a homodimer with higher in vitro activity towards noncanonical 5-fluoro-2'-deoxyuridine monophosphate than towards canonical thymidine monophosphate. Furthermore, we showed via in vivo assays that PynA protects cells against noncanonical pyrimidine derivatives such as 5-fluoro-2'-deoxyuridine and prevents the incorporation of the potentially mutagenic 5-bromo-2'-deoxyuridine (5-BrdU) into DNA. Fluctuation analysis performed under S. pneumoniae exposure to 5-BrdU revealed that the pynA null strain accumulates random mutations with high frequency, resulting in a 30-fold increase in the mutation rate. The data support a model in which PynA, a protein conserved in other Gram-positive bacteria, functions as a house-cleaning enzyme by selectively eliminating noncanonical nucleotides and maintaining the purity of dNTP pools, similar to the YjjG protein described for Escherichia coli.

Antibiotic Resistance in Czech Urban Wastewater Treatment Plants: Microbial and Molecular Genetic Characterization.

Quantitative changes in antibiotic resistance genes (ARGs) were investigated in six urban wastewater treatment plants (WWTPs) treating municipal and industrial wastewaters. In a selected WWTP, the fate of ARGs was studied in a 1-year time interval and in two phases of wastewater treatment process. Nine ARGs (tetW, tetO, tetA, tetB, tetM, blaTEM, ermB, sul1, and intl1) were quantified in total and their relative abundance assessed by ARG copies/16SrRNA copies. From the tetracycline resistance genes, tetW was the only one detected in all sampled WWTPs. Its relative abundance in the nitrification tank of WWTP5 was found stable during the 1-year period, but was lowered by secondary sedimentation processes in the wastewater treatment down to 24% compared to the nitrification tank. Bacterial isolates showing high tetracycline resistance (minimal inhibition concentrations >100 µg/mL) were identified as members of Acinetobacter, Klebsiella, Citrobacter, Bacillus, and Enterobacter genera. Dynamic shifts in the relative abundance of ermB and sul1 were also demonstrated in wastewater samples from WWTP5.

Allostery, Recognition of Nascent Peptidoglycan, and Cross-linking of the Cell Wall by the Essential Penicillin-Binding Protein 2x of Streptococcus pneumoniae.

Transpeptidases, members of the penicillin-binding protein (PBP) families, catalyze cross-linking of the bacterial cell wall. This transformation is critical for the survival of bacteria, and it is the target of inhibition by ß-lactam antibiotics. We report herein our structural insights into catalysis by the essential PBP2x of Streptococcus pneumoniae by disclosing a total of four X-ray structures, two computational models based on the crystal structures, and molecular-dynamics simulations. The X-ray structures are for the apo PBP2x, the enzyme modified covalently in the active site by oxacillin (a penicillin antibiotic), the enzyme modified by oxacillin in the presence of a synthetic tetrasaccharide surrogate for the cell-wall peptidoglycan, and a noncovalent complex of cefepime (a cephalosporin antibiotic) bound to the active site. A prerequisite for catalysis by transpeptidases, including PBP2x, is the molecular recognition of nascent peptidoglycan strands, which harbor pentapeptide stems. We disclose that the recognition of nascent peptidoglycan by PBP2x takes place by complexation of one pentapeptide stem at an allosteric site located in the PASTA domains of this enzyme. This binding predisposes the third pentapeptide stem in the same nascent peptidoglycan strand to penetration into the active site for the turnover events. The complexation of the two pentapeptide stems in the same peptidoglycan strand is a recognition motif for the nascent peptidoglycan, critical for the cell-wall cross-linking reaction.

A two-dimensional protein map of Pleurotus ostreatus microsomes-proteome dynamics.

Recent studies documented that several processes in filamentous fungi are connected with microsomal enzyme activities. In this work, microsomal subproteomes of Pleurotus ostreatus were analyzed by two-dimensional (2-D) polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. To assess proteome dynamics, microsomal proteins were isolated from fungal cultures after 7 and 12 days of cultivation. Additionally, 10 mg/L of 17α-ethinylestradiol (EE2) was treated with the cultures during 2 days. Despite the EE2 degradation by the fungus reached 97 and 76.3 % in 7- and 12-day-old cultures, respectively, only a minor effect on the composition of microsomal proteins was observed. The changes in protein maps related to ageing prevailed over those induced by EE2. Epoxide hydrolase, known to metabolize EE2, was detected in 12-day-old cultures only which suggests differences in EE2 degradation pathways utilized by fungal cultures of different age. The majority (32 %) of identified microsomal proteins were parts of mitochondrial energy metabolism.

Cytokines, growth, and environment factors in bone marrow plasma of acute lymphoblastic leukemia pediatric patients.

Acute lymphoblastic leukemia (ALL) cells depend on the microenvironment of the host in vivo and do not survive in in vitro culture. Conversely, the suppression of non-malignant tissues is one of the leading characteristics of the course of ALL. Both the non-malignant suppression and malignant cell survival may be partly affected by soluble factors within the bone marrow (BM) environment. Here, we aimed to identify proteins in BM plasma of children with ALL that may contribute to ALL aggressiveness and/or the microenvironment-mediated survival of ALL cells. LBMp (leukemic bone marrow plasma) at the time of ALL diagnosis was compared to control plasma of bone marrow (CBMp) or peripheral blood (CPBp) using a cytokine antibody array. The cytokine antibody array enabled simultaneous detection of 79 proteins per sample. Candidate proteins exhibiting significantly different profiles were further analyzed and confirmed by ELISA. mRNA expression of one of the candidate proteins (TIMP1) was studied using quantitative reverse transcriptase polymerase chain reaction (qRTPCR). The cytokine antibody array experiments identified 23 proteins that differed significantly (p<0.05); of these, two proteins (TIMP1 and LIF) withstood the Bonferroni correction. In contrast, little difference was observed between CBMp and CPBp. At the diagnosis of ALL, changes in the soluble microenvironment are detectable in BM plasma. These changes probably participate in the pathogenesis and/or result from the changes in the cell composition.

Surface hydrophobicity and roughness influences the morphology and biochemistry of streptomycetes during attached growth and differentiation.

Streptomycetes, soil-dwelling mycelial bacteria, can colonise surface of organic soil debris and soil particles. We analysed the effects of two different inert surfaces, glass and zirconia/silica, on the growth and antibiotic production in Streptomyces granaticolor. The surfaces used were in the form of microbeads and were surrounded by liquid growth media. Following the production of the antibiotic granaticin, more biomass was formed as well as a greater amount of antibiotic per milligram of protein on the glass beads than on the zirconia/silica beads. Comparison of young mycelium (6 h) proteomes, obtained from the cultures attached to the glass and zirconia/silica beads, revealed three proteins with altered expression levels (dihydrolipoamide dehydrogenase, amidophosphoribosyltransferase and cystathionine beta-synthase) and one unique protein (glyceraldehyde-3-phosphate dehydrogenase) that was present only in cells grown on glass beads. All of the identified proteins function primarily as cytoplasmic enzymes involved in different parts of metabolism; however, in several microorganisms, they are exposed on the cell surface and have been shown to be involved in adhesion or biofilm formation.

Fitness and proteome changes accompanying the development of erythromycin resistance in a population of Escherichia coli grown in continuous culture.

We studied the impact of a sublethal concentration of erythromycin on the fitness and proteome of a continuously cultivated population of Escherichia coli. The development of resistance to erythromycin in the population was followed over time by the gradient plate method and minimum inhibitory concentration (MIC) measurements. We measured the growth rate, standardized efficiency of synthesis of radiolabeled proteins, and translation accuracy of the system. The proteome changes were followed over time in two parallel experiments that differed in the presence or absence of erythromycin. A comparison of the proteomes at each time point (43, 68, and 103 h) revealed a group of unique proteins differing in expression. From all 35 proteins differing throughout the cultivation, only three were common to more than one time point. In the final population, a significant proportion of upregulated proteins was localized to the outer or inner cytoplasmic membranes or to the periplasmic space. In a population growing for more than 100 generations in the presence of antibiotic, erythromycin-resistant bacterial clones with improved fitness in comparison to early resistant culture predominated. This phenomenon was accompanied by distinct changes in protein expression during a stepwise, population-based development of erythromycin resistance.