Immunogenicity of novel vB_EcoS_NBD2 bacteriophage-originated nanotubes as a carrier for peptide-based vaccines.

Non-infectious virus-like nanoparticles mimic native virus structures and can be modified by inserting foreign protein fragments, making them immunogenic tools for antigen presentation. This study investigated, for the first time, the immunogenicity of long and flexible polytubes formed by yeast-expressed tail tube protein gp39 of bacteriophage vB_EcoS_NBD2 and evaluated their ability to elicit an immune response against the inserted protein fragments. Protein gp39-based polytubes induced humoral immune response in mice, even without the use of adjuvant. Bioinformatics analysis guided the selection of protein fragments from Acinetobacter baumannii for insertion into the C-terminus of gp39. Chimeric polytubes, displaying 28-amino acid long OmpA protein fragment, induced IgG response against OmpA protein fragment in immunized mice. These polytubes demonstrated their effectiveness both as antigen carrier and an adjuvant, when the OmpA fragments were either displayed on chimeric polytubes or used alongside with the unmodified polytubes. Our findings expand the potential applications of long and flexible polytubes, contributing to the development of novel antigen carriers with improved immunogenicity and antigen presentation capabilities.

Human nasal microbiota shifts in healthy and chronic respiratory disease conditions.

BACKGROUND: An increasing number of studies investigate various human microbiotas and their roles in the development of diseases, maintenance of health states, and balanced signaling towards the brain. Current data demonstrate that the nasal microbiota contains a unique and highly variable array of commensal bacteria and opportunistic pathogens. However, we need to understand how to harness current knowledge, enrich nasal microbiota with beneficial microorganisms, and prevent pathogenic developments. RESULTS: In this study, we have obtained nasal, nasopharyngeal, and bronchoalveolar lavage fluid samples from healthy volunteers and patients suffering from chronic respiratory tract diseases for full-length 16 S rRNA sequencing analysis using Oxford Nanopore Technologies. Demographic and clinical data were collected simultaneously. The microbiome analysis of 97 people from Lithuania suffering from chronic inflammatory respiratory tract disease and healthy volunteers revealed that the human nasal microbiome represents the microbiome of the upper airways well. CONCLUSIONS: The nasal microbiota of patients was enriched with opportunistic pathogens, which could be used as indicators of respiratory tract conditions. In addition, we observed that a healthy human nasal microbiome contained several plant- and bee-associated species, suggesting the possibility of enriching human nasal microbiota via such exposures when needed. These candidate probiotics should be investigated for their modulating effects on airway and lung epithelia, immunogenic properties, neurotransmitter content, and roles in maintaining respiratory health and nose-brain interrelationships.

Air pollution-associated shifts in the human airway microbiome and exposure-associated molecular events.

Publications addressing air pollution-induced human respiratory microbiome shifts are reviewed in this article. The healthy respiratory microbiota is characterized by a low density of bacteria, fungi and viruses with high diversity, and usually consists of Bacteroidetes, Firmicutes, Proteobacteria, Actinobacteria, Fusobacteria, viruses and fungi. The air's microbiome is highly dependent on air pollution levels and is directly reflected within the human respiratory microbiome. In addition, pollutants indirectly modify the local environment in human respiratory organs by reducing antioxidant capacity, misbalancing proteolysis and modulating inflammation, all of which regulate local microbiomes. Improving air quality leads to more diverse and healthy microbiomes of the local air and, subsequently, residents' airways.

The community of bacteria, viruses and fungi in the human body, known as the microbiome, plays an important role in human health. These communities vary in different locations in the body, for example in the gut, airways and skin. The microbiome within our airways is affected by air pollution because pollutants cause changes in the microbiome that may result in illness. In this article we review the available information on the effect of air pollution on the airway microbiome. We conclude that improving air quality is important to promoting healthy microbiomes and general human health.

A polyamine acetyltransferase regulates the motility and biofilm formation of Acinetobacter baumannii.

Acinetobacter baumannii is a nosocomial pathogen highly resistant to environmental changes and antimicrobial treatments. Regulation of cellular motility and biofilm formation is important for its virulence, although it is poorly described at the molecular level. It has been previously reported that Acinetobacter genus specifically produces a small positively charged metabolite, polyamine 1,3-diaminopropane, that has been associated with cell motility and virulence. Here we show that A. baumannii encodes novel acetyltransferase, Dpa, that acetylates 1,3-diaminopropane, directly affecting the bacterium motility. Expression of dpa increases in bacteria that form pellicle and adhere to eukaryotic cells as compared to planktonic bacterial cells, suggesting that cell motility is linked to the pool of non-modified 1,3-diaminopropane. Indeed, deletion of dpa hinders biofilm formation and increases twitching motion confirming the impact of balancing the levels of 1,3-diaminopropane on cell motility. The crystal structure of Dpa reveals topological and functional differences from other bacterial polyamine acetyltransferases, adopting a ß-swapped quaternary arrangement similar to that of eukaryotic polyamine acetyltransferases with a central size exclusion channel that sieves through the cellular polyamine pool. The structure of catalytically impaired DpaY128F in complex with the reaction product shows that binding and orientation of the polyamine substrates are conserved between different polyamine-acetyltransferases.

Novel Antibiotic Resistance Genes Identified by Functional Gene Library Screening in Stenotrophomonas maltophilia and Chryseobacterium spp. Bacteria of Soil Origin.

As one of the most diverse habitats of microorganisms, soil has been recognised as a reservoir of both antibiotics and the antibiotic resistance genes (ARGs). Bacteria naturally inhabiting soil or water often possess innate ARGs to counteract the chemical compounds produced by competitors living in the same environment. When such bacteria are able to cause infections in immunocompromised patients, their strong innate antibiotic resistance mechanisms make treatment difficult. We generated functional gene libraries using antibiotic-resistant Stenotrophomonas maltophilia and Chryseobacterium spp. bacteria isolated from agricultural soils in Lithuania to select for the genetic determinants responsible for their resistance. We were able to find novel variants of aminoglycoside and ß-lactam resistance genes, with ß-lactamases isolated from the Chryseobacterium spp. functional gene library, one of which is a variant of IND-like metallo-ß-lactamase (MBL) IND-17 and the other of which is a previously uncharacterised MBL we named CHM (Chryseobacterium metallo ß-lactamase). Our results indicate that soil microorganisms possess a diversity of ARG variants, which could potentially be transferred to the clinical setting.

The Impact of Intensive Fish Farming on Pond Sediment Microbiome and Antibiotic Resistance Gene Composition.

Aquaculture is a fast-growing animal food sector, and freshwater fish farming is particularly common in Central and Eastern Europe. As the biodiversity of fishery ponds is changed toward fulfilling the industrial needs, precautions should be taken to keep the system sustainable and protect the adjacent environment from possible damage. Due to risk of infectious diseases, antibiotics are used in aquaculture production systems. The constant exposure to antimicrobials can contribute to the rise of antibiotic resistance in aquaculture products and the adjacent ecosystems, with possibility of dissemination to the wider environment as well as between animals and humans. Even though previous studies have found antibiotic resistance genes in the sediments and water of farming ponds, the tendency and direction of spreading is not clear yet. The objective of this project was to evaluate the influence of intensive fish farming on the condition of water bodies used for the aquaculture and the environment, concentrating on the impact of the aquaculture on the surrounding water ecosystems as well as the possibility of transferring the pollutants and antibiotic resistance genes to both environment and the human hosts. Combined measurement of antibiotic and heavy metal contamination, toxicity assessment, microorganism diversity, and the detection of common antibiotic resistance genes was performed in the sediments of one fishery farm ponds as well as sampling points upstream and downstream. All the tested sediment samples did not show significantly elevated heavy metal concentrations and no substantial veterinary antibiotic pollution. From the antibiotic resistance genes tested, the presence of aminoglycoside and ß-lactam resistance determinants as well as the presence of integrons could be of concern for the possibility of transfer to humans. However, despite the lack of heavy metal and antibiotic pollution, the sediments showed toxicity, the cause of which should be explored more.

Microbial and Antimicrobial Resistance Profiles of Microbiota in Common Carps (Cyprinus carpio) from Aquacultured and Wild Fish Populations.

In this study we analyzed differences in microbial composition and antimicrobial resistance profiles in common carp living in two different environments: fish ponds, where carp have been kept under the same growing conditions over the last 50 years, and from the wild. The results demonstrated that wild fish carry a great variety of bacterial species (448 species with a prevalence of at least 0.01% from the total number of reads). Aquacultured individuals harbored 2.56 times fewer species in their gut. Significant microbial differences were observed in all taxonomic ranks, including bacterial classes and phyla. Besides bacterial variety, it was determined that aquacultured fish harbored more bacteria that are considered pathogens or opportunistic pathogens, such as Moraxellaceae, Flavobacteriaceae, and Staphylococcaceae. The frequency of antimicrobial resistance in bacterial indicators was more common in aquacultured fish than in wild fish, therefore fish farming may be a potential source of environmental contamination with antimicrobial resistant bacteria.

The Toxin-Antitoxin Systems of the Opportunistic Pathogen Stenotrophomonas maltophilia of Environmental and Clinical Origin.

Stenotrophomonas maltophilia is a ubiquitous environmental bacterium that has recently emerged as a multidrug-resistant opportunistic pathogen causing bloodstream, respiratory, and urinary tract infections. The connection between the commensal environmental S. maltophilia and the opportunistic pathogen strains is still under investigation. Bacterial toxin-antitoxin (TA) systems have been previously associated with pathogenic traits, such as biofilm formation and resistance to antibiotics, which are important in clinical settings. The same species of the bacterium can possess various sets of TAs, possibly influencing their overall stress response. While the TA systems of other important opportunistic pathogens have been researched, nothing is known about the TA systems of S. maltophilia. Here, we report the identification and characterization of S. maltophilia type II TA systems and their prevalence in the isolates of clinical and environmental origins. We found 49 putative TA systems by bioinformatic analysis in S. maltophilia genomes. Despite their even spread in sequenced S. maltophilia genomes, we observed that relBE, hicAB, and previously undescribed COG3832-ArsR operons were present solely in clinical S. maltophilia isolates collected in Lithuania, while hipBA was more frequent in the environmental ones. The kill-rescue experiments in Escherichia coli proved higBA, hicAB, and relBE systems to be functional TA modules. Together with different TA profiles, the clinical S. maltophilia isolates exhibited stronger biofilm formation, increased antibiotic, and serum resistance compared to environmental isolates. Such tendencies suggest that certain TA systems could be used as indicators of virulence traits.

Capsule Protects Acinetobacter baumannii From Inter-Bacterial Competition Mediated by CdiA Toxin.

Currently, Acinetobacter baumannii is considered as one of the most important infectious agents causing hospital acquired infections worldwide. It has been observed that many clinically important pathogens express contact-dependent growth inhibition (CDI) phenomenon, which modulates cell-cell and cell-environment interactions, potentially allowing bacteria to adapt to ever-changing conditions. Mainly, these systems are used for the inhibition of the growth of genetically different individuals within the same species. In this work, by performing cell competition assays with three genotypically different (as determined by pulse-field gel electrophoresis) clinical A. baumannii isolates II-c, II-a, and II-a1, we show that A. baumannii capsule is the main feature protecting from CDI-mediated inhibition. We also observed that for one clinical isolate, the two-component BfmRS system, contributed to the resistance against CDI-mediated inhibition. Moreover, we were able to demonstrate, that the effector protein CdiA is released into the growth media and exhibits its inhibitory activity without the requirement of a cell-cell contact. Lastly, by evaluating the remaining number of the cells pre-mixed with the CdiA and performing live/dead assay, we demonstrate that purified CdiA protein causes a rapid cell growth arrest. Our results indicate, that capsule efficiently protects A. baumannii from a CDI-mediated inhibition by a clinical A. baumannii V15 strain, which is able to secrete CdiA effector into the growth media and cause target cell growth arrest without a cell-cell contact.

Blp1 protein shows virulence-associated features and elicits protective immunity to Acinetobacter baumannii infection.

BACKGROUND: Multidrug resistant Acinetobacter baumannii is one of the major infection agents causing nosocomial pneumonia. Therefore, new therapeutic approaches against this bacterium are needed. Surface-exposed proteins from bacterial pathogens are implicated in a variety of virulence-related traits and are considered as promising candidates for vaccine development. RESULTS: We show in this study that a large Blp1 protein from opportunistic pathogen A. baumannii is encoded in all examined clinical strains of globally spread international clonal lineages I (IC I) and II (IC II). The two blp1 gene variants exhibit lineage-specific distribution profile. By characterization of blp1 deletion mutants and their complementation with blp1 alleles we show that blp1 gene is required for A. baumannii biofilm formation and adhesion to epithelial cells in IC I strain but not in the IC II strain. Nevertheless both alleles are functional in restoring the deficient phenotypes of IC I strain. Moreover, the blp1 gene is required for the establishing of A. baumannii virulence phenotype in nematode and murine infection models. Additionally, we demonstrate that C-terminal 711 amino acid fragment of Blp1 elicits an efficient protection to lethal A. baumannii infection in a murine model using active and passive immunization approaches. Antiserum obtained against Blp1-specific antigen provides opsonophagocytic killing of A. baumannii in vitro. CONCLUSIONS: Lineage-specific variants of surface-exposed components of bacterial pathogens complicate the development of new therapeutic approaches. Though we demonstrated different impact of Blp1 variants on adherence of IC I and IC II strains, Blp1-specific antiserum neutralized A. baumannii strains of both clonal lineages. Together with the observed increased survival rate in vaccinated mice these results indicate that A. baumannii Blp1 protein could be considered as a new vaccine candidate.

The role of Acinetobacter baumannii response regulator BfmR in pellicle formation and competitiveness via contact-dependent inhibition system.

BACKGROUND: Acinetobacter baumannii is one of the most important opportunistic pathogens responsible for hospital acquired infections. It displays multi-drug resistance profile and has the ability to colonize surfaces and persist under harsh conditions. A. baumannii two-component signal transduction system BfmRS, consisting of response regulator BfmR and sensor kinase BfmS, has been implicated in the control of various virulence-related traits and has been suggested to act as a global modulator of A. baumannii physiology. RESULTS: Here, we assessed the role of BfmR regulator in pellicle formation and bacterial competition, features important for the establishment of A. baumannii in clinical environment. We show that BfmR is required for the pellicle formation of A. baumannii, as ΔbfmRS mutant lacked this phenotype. The loss of bfmRS also greatly reduced the secretion of A. baumannii Hcp protein, which is a component of T6SS secretion system. However, T6SS-mediated killing phenotype was not impaired in ΔbfmRS mutant. On the contrary, the same mutation resulted in the transcriptional activation of contact-dependent inhibition (CDI) system, which A. baumannii used to inhibit the growth of another clinical A. baumannii strain and a closely related species Acinetobacter baylyi. CONCLUSIONS: The obtained results indicate that BfmR is not only required for the pellicle phenotype induction in A. baumannii, but also, due to the down-regulation of a CDI system, could allow the incorporation of other A. baumannii strains or related species, possibly increasing the likelihood of the pathogens' survival.

Microbial Diversity and Antimicrobial Resistance Profile in Microbiota From Soils of Conventional and Organic Farming Systems.

Soil is one of the biggest reservoirs of microbial diversity, yet the processes that define the community dynamics are not fully understood. Apart from soil management being vital for agricultural purposes, it is also considered a favorable environment for the evolution and development of antimicrobial resistance, which is due to its high complexity and ongoing competition between the microorganisms. Different approaches to agricultural production might have specific outcomes for soil microbial community composition and antibiotic resistance phenotype. Therefore in this study we aimed to compare the soil microbiota and its resistome in conventional and organic farming systems that are continually influenced by the different treatment (inorganic fertilizers and pesticides vs. organic manure and no chemical pest management). The comparison of the soil microbial communities revealed no major differences among the main phyla of bacteria between the two farming styles with similar soil structure and pH. Only small differences between the lower taxa could be observed indicating that the soil community is stable, with minor shifts in composition being able to handle the different styles of treatment and fertilization. It is still unclear what level of intensity can change microbial composition but current conventional farming in Central Europe demonstrates acceptable level of intensity for soil bacterial communities. When the resistome of the soils was assessed by screening the total soil DNA for clinically relevant and soil-derived antibiotic resistance genes, a low variety of resistance determinants was detected (resistance to ß-lactams, aminoglycosides, tetracycline, erythromycin, and rifampicin) with no clear preference for the soil farming type. The same soil samples were also used to isolate antibiotic resistant cultivable bacteria, which were predominated by highly resistant isolates of Pseudomonas, Stenotrophomonas, Sphingobacterium and Chryseobacterium genera. The resistance of these isolates was largely dependent on the efflux mechanisms, the soil Pseudomonas spp. relying mostly on RND, while Stenotrophomonas spp. and Chryseobacterium spp. on RND and ABC transporters.

The higBA Toxin-Antitoxin Module From the Opportunistic Pathogen Acinetobacter baumannii - Regulation, Activity, and Evolution.

Acinetobacter baumannii is one of the major causes of hard to treat multidrug-resistant hospital infections. A. baumannii features contributing to its spread and persistence in clinical environment are only beginning to be explored. Bacterial toxin-antitoxin (TA) systems are genetic loci shown to be involved in plasmid maintenance and proposed to function as components of stress response networks. Here we present a thorough characterization of type II system of A. baumannii, which is the most ubiquitous TA module present in A. baumannii plasmids. higBA of A. baumannii is a reverse TA (the toxin gene is the first in the operon) and shows little homology to other TA systems of RelE superfamily. It is represented by two variants, which both are functional albeit exhibit strong difference in sequence conservation. The higBA2 operon is found on ubiquitous 11 Kb pAB120 plasmid, conferring carbapenem resistance to clinical A. baumannii isolates and represents a higBA variant that can be found with multiple sequence variations. We show here that higBA2 is capable to confer maintenance of unstable plasmid in Acinetobacter species. HigB2 toxin functions as a ribonuclease and its activity is neutralized by HigA2 antitoxin through formation of an unusually large heterooligomeric complex. Based on the in vivo expression analysis of gfp reporter gene we propose that HigA2 antitoxin and HigBA2 protein complex bind the higBA2 promoter region to downregulate its transcription. We also demonstrate that higBA2 is a stress responsive locus, whose transcription changes in conditions encountered by A. baumannii in clinical environment and within the host. We show elevated expression of higBA2 during stationary phase, under iron deficiency and downregulated expression after antibiotic (rifampicin) treatment.

Surface-Related Features and Virulence Among Acinetobacter baumannii Clinical Isolates Belonging to International Clones I and II.

Acinetobacter baumannii currently represents one of the most important nosocomial infection agent due to its multidrug-resistance and a propensity for the epidemic spread. The A. baumannii strains belonging to the international clonal lineages I (IC I) and II (IC II) are associated with the hospital outbreaks and a high virulence. However, the intra and inter lineage-specific features of strains belonging to these most worldwide spread A. baumannii clones are not thoroughly explored. In this study we have investigated a set of cell surface-related features of A. baumannii IC I (n = 20) and IC II (n = 16) lineage strains, representing 30 distinct pulsed-field gel electrophoresis types in the collection of clinical isolates obtained in Lithuanian tertiary care hospitals. We show that A. baumannii IC II strains are non-motile, do not form pellicle and display distinct capsular polysaccharide profile compared with the IC I strains. Moreover, in contrast to the overall highly hydrophobic IC I strains, IC II strains showed a greater variation in cell surface hydrophobicity. Within the IC II lineage, hydrophilic strains demonstrated reduced ability to form biofilm and adhere to the abiotic surfaces, also possessed twofold thicker cell wall and exhibited higher resistance to desiccation. Furthermore, these strains showed increased adherence to the lung epithelial cells and were more virulent in nematode and mouse infection model compared with the hydrophobic IC II strains. According to the polymerase chain reaction-based locus-typing, the reduction in hydrophobicity of IC II strains was not capsule or lipooligosaccharide locus type-dependent. Hence, this study shows that the most widespread A. baumannii clonal lineages I and II markedly differ in the series of cell surface-related phenotypes including the considerable phenotypic diversification of IC II strains at the intra-lineage level. These findings suggest that the genotypically related A. baumannii strains might evolve the features which could provide an advantage at the specific conditions outside or within the host.

Another Brick in the Wall: a Rhamnan Polysaccharide Trapped inside Peptidoglycan of Lactococcus lactis.

Polysaccharides are ubiquitous components of the Gram-positive bacterial cell wall. In Lactococcus lactis, a polysaccharide pellicle (PSP) forms a layer at the cell surface. The PSP structure varies among lactococcal strains; in L. lactis MG1363, the PSP is composed of repeating hexasaccharide phosphate units. Here, we report the presence of an additional neutral polysaccharide in L. lactis MG1363 that is a rhamnan composed of α-l-Rha trisaccharide repeating units. This rhamnan is still present in mutants devoid of the PSP, indicating that its synthesis can occur independently of PSP synthesis. High-resolution magic-angle spinning nuclear magnetic resonance (HR-MAS NMR) analysis of whole bacterial cells identified a PSP at the surface of wild-type cells. In contrast, rhamnan was detected only at the surface of PSP-negative mutant cells, indicating that rhamnan is located underneath the surface-exposed PSP and is trapped inside peptidoglycan. The genetic determinants of rhamnan biosynthesis appear to be within the same genetic locus that encodes the PSP biosynthetic machinery, except the gene tagO encoding the initiating glycosyltransferase. We present a model of rhamnan biosynthesis based on an ABC transporter-dependent pathway. Conditional mutants producing reduced amounts of rhamnan exhibit strong morphological defects and impaired division, indicating that rhamnan is essential for normal growth and division. Finally, a mutation leading to reduced expression of lcpA, encoding a protein of the LytR-CpsA-Psr (LCP) family, was shown to severely affect cell wall structure. In lcpA mutant cells, in contrast to wild-type cells, rhamnan was detected by HR-MAS NMR, suggesting that LcpA participates in the attachment of rhamnan to peptidoglycan.IMPORTANCE In the cell wall of Gram-positive bacteria, the peptidoglycan sacculus is considered the major structural component, maintaining cell shape and integrity. It is decorated with other glycopolymers, including polysaccharides, the roles of which are not fully elucidated. In the ovococcus Lactococcus lactis, a polysaccharide with a different structure between strains forms a layer at the bacterial surface and acts as the receptor for various bacteriophages that typically exhibit a narrow host range. The present report describes the identification of a novel polysaccharide in the L. lactis cell wall, a rhamnan that is trapped inside the peptidoglycan and covalently bound to it. We propose a model of rhamnan synthesis based on an ABC transporter-dependent pathway. Rhamnan appears as a conserved component of the lactococcal cell wall playing an essential role in growth and division, thus highlighting the importance of polysaccharides in the cell wall integrity of Gram-positive ovococci.

Regulation of Cell Wall Plasticity by Nucleotide Metabolism in Lactococcus lactis.

To ensure optimal cell growth and separation and to adapt to environmental parameters, bacteria have to maintain a balance between cell wall (CW) rigidity and flexibility. This can be achieved by a concerted action of peptidoglycan (PG) hydrolases and PG-synthesizing/modifying enzymes. In a search for new regulatory mechanisms responsible for the maintenance of this equilibrium in Lactococcus lactis, we isolated mutants that are resistant to the PG hydrolase lysozyme. We found that 14% of the causative mutations were mapped in the guaA gene, the product of which is involved in purine metabolism. Genetic and transcriptional analyses combined with PG structure determination of the guaA mutant enabled us to reveal the pivotal role of the pyrB gene in the regulation of CW rigidity. Our results indicate that conversion of l-aspartate (l-Asp) to N-carbamoyl-l-aspartate by PyrB may reduce the amount of l-Asp available for PG synthesis and thus cause the appearance of Asp/Asn-less stem peptides in PG. Such stem peptides do not form PG cross-bridges, resulting in a decrease in PG cross-linking and, consequently, reduced PG thickness and rigidity. We hypothesize that the concurrent utilization of l-Asp for pyrimidine and PG synthesis may be part of the regulatory scheme, ensuring CW flexibility during exponential growth and rigidity in stationary phase. The fact that l-Asp availability is dependent on nucleotide metabolism, which is tightly regulated in accordance with the growth rate, provides L. lactis cells the means to ensure optimal CW plasticity without the need to control the expression of PG synthesis genes.

Surface proteome analysis of a natural isolate of Lactococcus lactis reveals the presence of pili able to bind human intestinal epithelial cells.

Surface proteins of Gram-positive bacteria play crucial roles in bacterial adhesion to host tissues. Regarding commensal or probiotic bacteria, adhesion to intestinal mucosa may promote their persistence in the gastro-intestinal tract and their beneficial effects to the host. In this study, seven Lactococcus lactis strains exhibiting variable surface physico-chemical properties were compared for their adhesion to Caco-2 intestinal epithelial cells. In this test, only one vegetal isolate TIL448 expressed a high-adhesion phenotype. A nonadhesive derivative was obtained by plasmid curing from TIL448, indicating that the adhesion determinants were plasmid-encoded. Surface-exposed proteins in TIL448 were analyzed by a proteomic approach consisting in shaving of the bacterial surface with trypsin and analysis of the released peptides by LC-MS/MS. As the TIL448 complete genome sequence was not available, the tryptic peptides were identified by a mass matching approach against a database including all Lactococcus protein sequences and the sequences deduced from partial DNA sequences of the TIL448 plasmids. Two surface proteins, encoded by plasmids in TIL448, were identified as candidate adhesins, the first one displaying pilin characteristics and the second one containing two mucus-binding domains. Inactivation of the pilin gene abolished adhesion to Caco-2 cells whereas inactivation of the mucus-binding protein gene had no effect on adhesion. The pilin gene is located inside a cluster of four genes encoding two other pilin-like proteins and one class-C sortase. Synthesis of pili was confirmed by immunoblotting detection of high molecular weight forms of pilins associated to the cell wall as well as by electron and atomic force microscopy observations. As a conclusion, surface proteome analysis allowed us to detect pilins at the surface of L. lactis TIL448. Moreover we showed that pili appendages are formed and involved in adhesion to Caco-2 intestinal epithelial cells.

Characterization of Escherichia coli dinJ-yafQ toxin-antitoxin system using insights from mutagenesis data.

Escherichia coli dinJ-yafQ operon codes for a functional toxin-antitoxin (TA) system. YafQ toxin is an RNase which, upon overproduction, specifically inhibits the translation process by cleaving cellular mRNA at specific sequences. DinJ is an antitoxin and counteracts YafQ-mediated toxicity by forming a strong protein complex. In the present study we used site-directed mutagenesis of YafQ to determine the amino acids important for its catalytic activity. His50Ala, His63Ala, Asp67Ala, Trp68Ala, Trp68Phe, Arg83Ala, His87Ala, and Phe91Ala substitutions of the predicted active-site residues of YafQ abolished mRNA cleavage in vivo, whereas Asp61Ala and Phe91Tyr mutations inhibited YafQ RNase activity only moderately. We show that YafQ, upon overexpression, cleaved mRNAs preferably 5' to A between the second and third nucleotides in the codon in vivo. YafQ also showed RNase activity against mRNA, tRNA, and 5S rRNA molecules in vitro, albeit with no strong specificity. The endoribonuclease activity of YafQ was inhibited in the complex with DinJ antitoxin in vitro. DinJ-YafQ protein complex and DinJ antitoxin alone selectively bind to one of the two palindromic sequences present in the intergenic region upstream of the dinJ-yafQ operon, suggesting the autoregulation mode of this TA system.

Tigecycline - how powerful is it in the fight against antibiotic-resistant bacteria?

Tigecycline is a semisynthetic analogue of earlier tetracyclines and represents the first member of a novel class of antimicrobials - glycylcyclines - recently approved for clinical use. It is active against a broad range of gram-negative and gram-positive bacterial species including clinically important multidrug-resistant nosocomial and community-acquired bacterial pathogens. The exact molecular basis of tigecycline action is not clear at present, although similarly to the tetracyclines, it has been shown to inhibit the translation elongation step by binding to the ribosome 30S subunit and preventing aminoacylated tRNAs to accommodate in the ribosomal A site. Importantly, tigecycline overcomes the action of ribosomal protection proteins and is not a substrate for tetracycline efflux pumps of most bacteria - well-known and prevalent cellular mechanisms of microbial tetracycline resistance. The present review summarizes current knowledge on the molecular mechanism of the tigecycline action, antibacterial activity against various bacteria, clinical application, development of resistance to glycylcyclines.

An Escherichia coli asr mutant has decreased fitness during colonization in a mouse model.

The Escherichia coli asr gene, like its homologues in other enterobacteria, is strongly induced by low external pH. The E. coli asr mutant shows weakened ability to adapt to acidic pH. This suggests that the asr gene product is important for enterobacterial species, both commensal and pathogenic, in overcoming acid stress in the stomach and subsequently colonizing the intestine. We examined the relative fitness of an E. coli asr mutant compared to a wild type, by feeding both strains simultaneously to mice and letting them colonize the intestine. Analysis of the bacteria after passage through the intestine showed up to five orders of magnitude less asr mutant than wild type. Transcomplementation of the asr gene on a plasmid partially restored the number of mutants. Similar competition in liquid media demonstrated that the asr mutant has reduced viability during long-term incubation in rich media, but is as fit as the wild type when bacteria are challenged in minimal medium. Competition carried out under different pH conditions proved that pH of the media was not the main determinant leading to the decreased fitness of the asr mutant. This suggests that the asr gene product is important for adaptation to stress conditions other than acidity, including long periods of starvation.