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Mycobacterium phage Adephagia is a cluster K phage that infects Mycobacterium smegmatis and some strains of Mycobacterium pathogens. Adephagia has a siphoviral virion morphology and is temperate. Its genome is 59,646 bp long and codes for one tRNA gene and 94 predicted protein-coding genes; most genes not associated with virion structure and assembly are functionally ill-defined. Here, we determined the Adephagia gene expression patterns in lytic and lysogenic growth and used structural predictions to assign additional gene functions. We characterized 66 nonstructural genes for their toxic phenotypes when expressed in M. smegmatis , and we show that 25 of these (38%) are either toxic or strongly inhibit growth, resulting in either reduced viability or small colony sizes. Some of these genes are predicted to be involved in DNA metabolism or regulation, but others are of unknown function. We also characterize the HicAB-like toxin–antitoxin (TA) system encoded by Adephagia (gp91 and gp90, respectively) and show that the gp90 antitoxin is lysogenically expressed, abrogates gp91 toxicity, and is required for normal lytic and lysogenic growth. [ABSTRACT FROM AUTHOR]

Bacterial ubiquitous Toxin-Antitoxin (TA) systems are considered to be important survival mechanisms during stress conditions. In regular environmental conditions, the antitoxin blocks the toxin, whereas during imbalanced conditions, the antitoxin concentration decreases, exposing the bacteria cell to a range of toxic events. The most evident consequence of this disequilibrium is cell growth arrest, which is the reason why TAs are generally described as active in the function of bacterial growth kinetics. Virulence-associated proteins B and C (VapBC) are a family of type II TA system, in which VapC is predicted to display the toxic ribonuclease activity while VapB counteracts this activity. Previously, using in silico data, we designated four VapBC TA modules in Leptospira interrogans serovar Copenhageni, the main etiological agent of human leptospirosis in Brazil. The present study aimed to obtain the proteins and functionally characterize the VapBC-1 module. The expression of the toxin gene vapC in E. coli did not decrease the cell growth rate in broth culture, as was expected to happen within active TA modules. However, interestingly, when the expression of the toxin was compared to that of the complexed toxin and antitoxin, cell viability was strongly affected, with a decrease of three orders of magnitude in colony forming unity (CFU). The assumption of the affinity between the toxin and the antitoxin was confirmed in vivo through the observation of their co-purification from cultivation of E. coli co-expressing vapB-vapC genes. RNAse activity assays showed that VapC-1 cleaves MS2 RNA and ribosomal RNA from L. interrogans. Our results indicate that the VapBC-1 module is a potentially functional TA system acting on targets that involve specific functions. It is very important to emphasize that the common attribution of the functionality of TA modules cannot be defined based merely on their ability to inhibit bacterial growth in a liquid medium. [ABSTRACT FROM AUTHOR]

Type I toxin-antitoxin systems (T1TAs) are bipartite bacterial loci encoding a growth-inhibitory toxin and an antitoxin small RNA (sRNA). In many of these systems, the transcribed toxin mRNA is translationally inactive, but becomes translation-competent upon ribonucleolytic processing. The antitoxin sRNA targets the processed mRNA to inhibit its translation. This two-level control mechanism prevents cotranscriptional translation of the toxin and allows its synthesis only when the antitoxin is absent. Contrary to this, we found that the timP mRNA of the timPR T1TA locus does not undergo enzymatic processing. Instead, the full-length timP transcript is both translationally active and can be targeted by the antitoxin TimR. Thus, tight control in this system relies on a noncanonical mechanism. Based on the results from in vitro binding assays, RNA structure probing, and cell-free translation experiments, we suggest that timP mRNA adopts mutually exclusive structural conformations. The active form uniquely possesses an RNA pseudoknot structure which is essential for translation initiation. TimR preferentially binds to the active conformation, which leads to pseudoknot destabilization and inhibited translation. Based on this, we propose a model in which "structural processing" of timP mRNA enables tight inhibition by TimR in nonpermissive conditions, and TimP synthesis only upon TimR depletion. [ABSTRACT FROM AUTHOR]

Toxin-antitoxin (TA) systems are the major mechanism for persister formation in Mycobacterium tuberculosis (Mtb). Previous studies found that HigBA2 (Rv2022c-Rv2021c), a predicted type II TA system of Mtb, could be activated for transcription in response to multiple stresses such as anti-tuberculosis drugs, nutrient starvation, endure hypoxia, acidic pH, etc. In this study, we determined the binding site of HigA2 (Rv2021c), which is located in the coding region of the upstream gene higB2 (Rv2022c), and the conserved recognition motif of HigA2 was characterized via oligonucleotide mutation. Eight binding sites of HigA2 were further found in the Mtb genome according to the conserved motif. RT-PCR showed that HigA2 can regulate the transcription level of all eight of these genes and three adjacent downstream genes. DNA pull-down experiments showed that twelve functional regulators sense external regulatory signals and may regulate the transcription of the HigBA2 system. Of these, Rv0903c, Rv0744c, Rv0474, Rv3124, Rv2603c, and Rv3583c may be involved in the regulation of external stress signals. In general, we identified the downstream target genes and possible upstream regulatory genes of HigA2, which paved the way for the illustration of the persistence establishment mechanism in Mtb. [ABSTRACT FROM AUTHOR]

Mechanistic understanding of antibiotic persistence is a prerequisite in controlling the emergence of MDR cases in Tuberculosis (TB). We have reported that the cholesterol-induced activation of VapC12 ribonuclease is critical for disease persistence in TB. In this study, we observed that relative to the wild type, mice infected with DvapC12 induced a pro-inflammatory response, had a higher pathogen load, and responded better to the anti-TB treatment. In a high-dose infection model, all the mice infected with DvapC12 succumbed early to the disease. Finally, we reported that the above phenotype of DvapC12 was dependent on the presence of the TLR4 receptor. Overall, the data suggests that failure of a timely resolution of the early inflammation by the DvapC12 infected mice led to hyperinflammation, altered T-cell response and high bacterial load. In conclusion, our findings suggest the role of the VapC12 toxin in modulating the innate immune response of the host in ways that favor the long-term survival of the pathogen inside the host. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (M. tb) employs an extensive network of more than 90 toxin–antitoxin systems, and among them, VapC toxins are the most abundant. While most VapCs function as classical RNases with toxic effects, a significant number of them do not exhibit toxicity. However, these non-toxic VapCs may retain specific RNA binding abilities as seen in case of VapC16, leading to ribosome stalling at specific codons and reprofiling M. tb's proteome to aid in the bacterium's survival under different stressful conditions within the host. Here, we challenge the conventional classification of all VapC toxins as RNases and highlight the complexity of M. tb's strategies for survival and adaptation during infection. [ABSTRACT FROM AUTHOR]

ABSTRACT Burkholderia pseudomallei (Bpm) is a Gram-negative intracellular pathogen that causes melioidosis in humans, a neglected, underreported, and lethal disease that can reach a fatal outcome in over 50% of the cases. It can produce both acute and chronic infections, the latter being particularly challenging to eliminate because of the intracellular life cycle of the bacteria and its ability to generate a “persister” dormant state. The molecular mechanism that allows the switch between growing and persister phenotypes is not well understood but it is hypothesized to be due at least in part to the participation of toxin-antitoxin (TA) systems. We have previously studied the link between one of those systems (defined as HigBA) with specific expression patterns associated with levofloxacin antibiotic exposure. Through in silico methods, we predicted the presence of another three pairs of genes encoding for additional putative HigBA systems. Therefore, our main goal was to establish which mechanisms are conserved as well as which pathways are specific among different Bpm TA systems from the same family. We hypothesize that the high prevalence, and sometimes even redundancy of these systems in the Bpm chromosomes indicates that they can interact with each other and not function as only individual systems, as it was traditionally thought, and might be playing an undefined role in Bpm lifecycle. Here, we show that both the toxin and the antitoxin of the different systems contribute to bacterial survival and that toxins from the same family can have a cumulative effect under environmental stressful conditions.IMPORTANCEToxin-antitoxin (TA) systems play a significant role in bacterial persistence, a phenomenon where bacterial cells enter a dormant or slow-growing state to survive adverse conditions such as nutrient deprivation, antibiotic exposure, or host immune responses. By studying TA systems in Burkholderia pseudomallei, we can gain insights into how this pathogen survives and persists in the host environment, contributing to its virulence and ability to cause melioidosis chronic infections.

ABSTRACT Bacterial chromosomal type I toxin-antitoxin systems consist of a small protein, typically under 60 amino acids, and a small RNA (sRNA) that represses toxin translation. These gene pairs have gained attention over the last decade for their contribution to antibiotic persistence and phage tolerance in bacteria. However, biological functions for many remain elusive as gene deletions often fail to produce an observable phenotype. For many pairs, it is still unknown when the toxin and/or antitoxin gene are natively expressed within the bacterium. We examined sequence conservation of three type I toxin-antitoxin systems, tisB/istR-1, shoB/ohsC, and zor/orz, in over 2,000 Escherichia coli strains, including pathogenic and commensal isolates. Using our custom database, we found that these gene pairs are widespread across E. coli and have expression potential via BLASTn. We identified an alternative, dominant sequence variant of TisB and confirmed that it is toxic upon overproduction. Additionally, analyses revealed a highly conserved sequence in the zorO mRNA untranslated region that is required for full toxicity. We further noted that over 30% of E. coli genomes contain an orz antitoxin gene only and confirmed its expression in a representative strain: the first confirmed report of a type I antitoxin without its cognate toxin. Our results add to our understanding of these systems, and our methodology is applicable for other type I loci to identify critical regulatory and functional features.IMPORTANCEChromosomal type I toxin-antitoxins are a class of genes that have gained increasing attention over the last decade for their roles in antibiotic persistence which may contribute to therapeutic failures. However, the control of many of these genes and when they function have remained elusive. We demonstrate that a simple genetic conservation-based approach utilizing free, publicly available data yields known and novel insights into the regulation and function of three chromosomal type I toxin-antitoxins in Escherichia coli. This study also provides a framework for how this approach could be applied to other genes of interest.

Bacterial toxin-antitoxin (TA) systems are widespread in chromosomes and plasmids of free-living microorganisms, but only a few have been identified in obligate intracellular species. We found seven putative type II TA modules in Waddlia chondrophila, a Chlamydia-related species that is able to infect a very broad series of eukaryotic hosts, ranging from protists to mammalian cells. The RNA levels of Waddlia TA systems are significantly upregulated by iron starvation and novobiocin, but they are not affected by antibiotics such as ß-lactams and glycopeptides, which suggests different mechanisms underlying stress responses. Five of the identified TA modules, including HigBA1 and MazEF1, encoded on the Waddlia cryptic plasmid, proved to be functional when expressed in a heterologous host. TA systems have been associated with the maintenance of mobile genetic elements, bacterial defense against bacteriophages, and persistence upon exposure to adverse conditions. As their RNA levels are upregulated upon exposure to adverse conditions, Waddlia TA modules may be involved in survival to stress. Moreover, as Waddlia can infect a wide range of hosts including free-living amoebae, TA modules could also represent an innate immunity system to fight against bacteriophages and other microorganisms with which Waddlia has to share its replicative niche. [ABSTRACT FROM AUTHOR]

Mechanistic understanding of antibiotic persistence is a prerequisite in controlling the emergence of MDR cases in Tuberculosis (TB). We have reported that the cholesterol-induced activation of VapC12 ribonuclease is critical for disease persistence in TB. In this study, we observed that relative to the wild type, mice infected with ΔvapC12 induced a pro-inflammatory response, had a higher pathogen load, and responded better to the anti-TB treatment. In a high-dose infection model, all the mice infected with ΔvapC12 succumbed early to the disease. Finally, we reported that the above phenotype of ΔvapC12 was dependent on the presence of the TLR4 receptor. Overall, the data suggests that failure of a timely resolution of the early inflammation by the ΔvapC12 infected mice led to hyperinflammation, altered T-cell response and high bacterial load. In conclusion, our findings suggest the role of the VapC12 toxin in modulating the innate immune response of the host in ways that favor the long-term survival of the pathogen inside the host.

Bacterial ubiquitous Toxin-Antitoxin (TA) systems are considered to be important survival mechanisms during stress conditions. In regular environmental conditions, the antitoxin blocks the toxin, whereas during imbalanced conditions, the antitoxin concentration decreases, exposing the bacteria cell to a range of toxic events. The most evident consequence of this disequilibrium is cell growth arrest, which is the reason why TAs are generally described as active in the function of bacterial growth kinetics. Virulence-associated proteins B and C (VapBC) are a family of type II TA system, in which VapC is predicted to display the toxic ribonuclease activity while VapB counteracts this activity. Previously, using in silico data, we designated four VapBC TA modules in Leptospira interrogans serovar Copenhageni, the main etiological agent of human leptospirosis in Brazil. The present study aimed to obtain the proteins and functionally characterize the VapBC-1 module. The expression of the toxin gene vapC in E. coli did not decrease the cell growth rate in broth culture, as was expected to happen within active TA modules. However, interestingly, when the expression of the toxin was compared to that of the complexed toxin and antitoxin, cell viability was strongly affected, with a decrease of three orders of magnitude in colony forming unity (CFU). The assumption of the affinity between the toxin and the antitoxin was confirmed in vivo through the observation of their co-purification from cultivation of E. coli co-expressing vapB-vapC genes. RNAse activity assays showed that VapC-1 cleaves MS2 RNA and ribosomal RNA from L. interrogans. Our results indicate that the VapBC-1 module is a potentially functional TA system acting on targets that involve specific functions. It is very important to emphasize that the common attribution of the functionality of TA modules cannot be defined based merely on their ability to inhibit bacterial growth in a liquid medium.

Toxin-antitoxin (TA) systems are the major mechanism for persister formation in Mycobacterium tuberculosis (Mtb). Previous studies found that HigBA2 (Rv2022c-Rv2021c), a predicted type II TA system of Mtb, could be activated for transcription in response to multiple stresses such as anti-tuberculosis drugs, nutrient starvation, endure hypoxia, acidic pH, etc. In this study, we determined the binding site of HigA2 (Rv2021c), which is located in the coding region of the upstream gene higB2 (Rv2022c), and the conserved recognition motif of HigA2 was characterized via oligonucleotide mutation. Eight binding sites of HigA2 were further found in the Mtb genome according to the conserved motif. RT-PCR showed that HigA2 can regulate the transcription level of all eight of these genes and three adjacent downstream genes. DNA pull-down experiments showed that twelve functional regulators sense external regulatory signals and may regulate the transcription of the HigBA2 system. Of these, Rv0903c, Rv0744c, Rv0474, Rv3124, Rv2603c, and Rv3583c may be involved in the regulation of external stress signals. In general, we identified the downstream target genes and possible upstream regulatory genes of HigA2, which paved the way for the illustration of the persistence establishment mechanism in Mtb.

ABSTRACT Toxin-antitoxin (TA) systems are widespread among prokaryotes and can play an important role in the maintenance of genetic elements and pathogen virulence. Their roles in bacterial stress response and antimicrobial persistence currently remain highly controversial. The current study investigated a novel TA system (Aave_1720-Aave_1719) from the phytopathogenic bacterium Acidovorax citrulli, which had characteristics of both type II and type V TA systems. Heterologous expression in Escherichia coli confirmed that Aave_1720 functions as a toxin gene, while Aave_1719 acts as antitoxin gene. The Aave_1720 toxin functions as a ribonuclease, and antitoxin Aave_1719 binds directly to Aave_1720 to inhibit its ribonuclease activity, which is typical of type II TA systems. Interestingly, Aave_1719 itself was found to exhibit ribonuclease activity with a preference for targeting Aave_1720 mRNA, which indicates that it can also neutralize the Aave_1720 toxin in a manner characteristic of type V TA systems. Expression analysis revealed that the Aave_1719 gene is upregulated in response to sodium hypochlorite stress and that deletion of the Aave_1720 gene individually or in combination with Aave_1719 affected biofilm formation, as well as the survival of A. citrulli in response to sodium hypochlorite stress. Taken together, these findings expand our understanding of TA systems revealing a novel mechanism of toxin neutralization that combines aspects of both type II and type V TA systems. IMPORTANCE Bacterial fruit blotch (BFB), which is caused by the seed-borne bacterium Acidovorax citrulli, is a devastating disease affecting cucurbit crops throughout the world. Although seed fermentation and treatment with disinfectants can provide effective management of BFB, they cannot completely guarantee pathogen-free seedstock, which suggests that A. citrulli is a highly stress-resistant pathogen. Toxin-antitoxin (TA) systems are common among a diverse range of bacteria and have been reported to play a role in bacterial stress response. However, there is currently much debate about the relationship between TA systems and stress response in bacteria. The current study characterized a novel TA system (Aave_1720-Aave_1719) from A. citrulli that affects both biofilm formation and survival in response to sodium hypochlorite stress. The mechanism of neutralization differed from typical TA systems as two separate mechanisms were associated with the antitoxin, which exhibited characteristics of both type II and type V TA systems. The Aave_1720-Aave_1719 system described here also constitutes the first known report of a double-ribonuclease TA system in bacteria, which expands our understanding of the range of regulatory mechanisms utilized by bacterial TA systems, providing new insight into the survival of A. citrulli in response to stress.

Introduction: Non-tuberculous mycobacteria (NTM) is a major category of environmental bacteria in nature that can be divided into rapidly growing mycobacteria (RGM) and slowly growing mycobacteria (SGM) based on their distinct growth rates. To explore differential molecular mechanisms between RGM and SGM is crucial to understand their survival state, environmental/host adaptation and pathogenicity. Comparative genomic analysis provides a powerful tool for deeply investigating differential molecular mechanisms between them. However, large- scale comparative genomic analysis between RGM and SGM is still uncovered. Methods: In this study, we screened 335 high-quality, non-redundant NTM genome sequences covering 187 species from 3,478 online NTM genomes, and then performed a comprehensive comparative genomic analysis to identify differential genomic characteristics and featured genes/protein domains between RGM and SGM. Results: Our findings reveal that RGM has a larger genome size, more genes, lower GC content, and more featured genes/protein domains in metabolism of some main substances (e.g. carbohydrates, amino acids, nucleotides, ions, and coenzymes), energy metabolism, signal transduction, replication, transcription, and translation processes, which are essential for its rapid growth requirements. On the other hand, SGM has a smaller genome size, fewer genes, higher GC content, and more featured genes/protein domains in lipid and secondary metabolite metabolisms and cellular defense mechanisms, which help enhance its genome stability and environmental adaptability. Additionally, orthogroup analysis revealed the important roles of bacterial division and bacteriophage associated genes in RGM and secretion system related genes for better environmental adaptation in SGM. Notably, PCoA analysis of the top 20 genes/protein domains showed precision classification between RGM and SGM, indicating the credibility of our screening/classification strategies. Discussion: Overall, our findings shed light on differential underlying molecular mechanisms in survival state, adaptation and pathogenicity between RGM and SGM, show the potential for our comparative genomic pipeline to investigate differential genes/protein domains at whole genomic level across different bacterial species on a large scale, and provide an important reference and improved understanding of NTM. [ABSTRACT FROM AUTHOR]

Recombination-promoting nuclease (Rpn) proteins are broadly distributed across bacterial phyla, yet their functions remain unclear. Here, we report that these proteins are toxin-antitoxin systems, comprised of genes-within-genes, that combat phage infection. We show the small, highly variable Rpn C-terminal domains (RpnS), which are translated separately from the full-length proteins (RpnL), directly block the activities of the toxic RpnL. The crystal structure of RpnAS revealed a dimerization interface encompassing a helix that can have four amino acid repeats whose number varies widely among strains of the same species. Consistent with strong selection for the variation, we document that plasmid-encoded RpnP2L protects Escherichia coli against certain phages. We propose that many more intragenic-encoded proteins that serve regulatory roles remain to be discovered in all organisms. [ABSTRACT FROM AUTHOR]

Toxin-antitoxin (TA) systems are widely present in bacterial genomes. They consist of stable toxins and unstable antitoxins that are classified into distinct groups based on their structure and biological activity. TA systems are mostly related to mobile genetic elements and can be easily acquired through horizontal gene transfer. The ubiquity of different homologous and non-homologous TA systems within a single bacterial genome raises questions about their potential cross-interactions. Unspecific cross-talk between toxins and antitoxins of non-cognate modules may unbalance the ratio of the interacting partners and cause an increase in the free toxin level, which can be deleterious to the cell. Moreover, TA systems can be involved in broadly understood molecular networks as transcriptional regulators of other genes' expression or modulators of cellular mRNA stability. In nature, multiple copies of highly similar or identical TA systems are rather infrequent and probably represent a transition stage during evolution to complete insulation or decay of one of them. Nevertheless, several types of cross-interactions have been described in the literature to date. This implies a question of the possibility and consequences of the TA system cross-interactions, especially in the context of the practical application of the TA-based biotechnological and medical strategies, in which such TAs will be used outside their natural context, will be artificially introduced and induced in the new hosts. Thus, in this review, we discuss the prospective challenges of system cross-talks in the safety and effectiveness of TA system usage. [ABSTRACT FROM AUTHOR]

The study of fusobacterial virulence factors has dramatically benefited from the creation of various genetic tools for DNA manipulation, including galK-based counterselection for in-frame deletion mutagenesis in Fusobacterium nucleatum, which was recently developed. However, this method requires a host lacking the galK gene, which is an inherent limitation. To circumvent this limitation, we explored the possibility of using the hicA gene that encodes a toxin consisting of a HicAB toxin-antitoxin module in Fusobacterium periodonticum as a new counterselective marker. Interestingly, the full-length hicA gene is not toxic in F. nucleatum, but a truncated hicA gene version lacking the first six amino acids is functional as a toxin. The toxin expression is driven by an rpsJ promoter and is controlled at its translational level by using a theophylline-responsive riboswitch unit. As a proof of concept, we created markerless in-frame deletions in the fusobacterial adhesin radD gene within the F. nucleatum rad operon and the tnaA gene that encodes the tryptophanase for indole production. After vector integration, plasmid excision after counterselection appeared to have occurred in 100% of colonies grown on theophylline-added plates and resulted in in-frame deletions in 50% of the screened isolates. This hicA-based counterselection system provides a robust and reliable counterselection in wild-type background F. nucleatum and should also be adapted for use in other bacteria. IMPORTANCE: Fusobacterium nucleatum is an indole-producing human oral anaerobe associated with periodontal diseases, preterm birth, and several cancers. Little is known about the mechanisms of fusobacterial pathogenesis and associated factors, mainly due to the lack of robust genetic tools for this organism. Here, we showed that a mutated hicA gene from Fusobacterium periodonticum expresses an active toxin and was used as a counterselection marker. This hicA-based in-frame deletion system efficiently creates in-frame deletion mutations in the wild-type background of F. nucleatum. This is the first report to use the hicA gene as a counterselection marker in a bacterial genetic study. [ABSTRACT FROM AUTHOR]

ABSTRACT The Streptococcus mutans genetic system offers a variety of strategies to rapidly engineer targeted chromosomal mutations. Previously, we reported the first S. mutans negative selection system that functions in a wild-type background. This system utilizes induced sensitivity to the toxic amino acid analog p-chlorophenylalanine (4-CP) as a negative selection mechanism and was developed for counterselection-based cloning-independent markerless mutagenesis (CIMM). While we have employed this system extensively for our ongoing genetic studies, we have encountered a couple limitations with the system, mainly its narrow host range and the requirement for selection on a toxic substrate. Here, we report the development of a new negative selection system that addresses both limitations, while still retaining the utility of the previous 4-CP-based markerless mutagenesis system. We placed a variety of toxin-encoding genes under the control of the xylose-inducible gene expression cassette (Xyl-S) and found the Fst-sm and ParE toxins to be suitable candidates for inducible negative selection. We combined the inducible toxins with an antibiotic resistance gene to create several different counterselection cassettes. The most broadly useful of these contained a wild-type fst-sm open reading frame transcriptionally fused to a point mutant form of the Xyl-S expression system, which we subsequently named IFDC4. IFDC4 was shown to exhibit exceptionally low background resistance, with 3- to 4-log reductions in cell number observed when plating on xylose-supplemented medium. IFDC4 also functioned similarly in multiple strains of S. mutans as well as with Streptococcus gordonii and Streptococcus sanguinis. We performed CIMM with IFDC4 and successfully engineered a variety of different types of markerless mutations in all three species. The counterselection strategy described here provides a template approach that should be adaptable for the creation of similar counterselection systems in many other bacteria. IMPORTANCE Multiple medically significant Streptococcus species, such as S. mutans, have highly sophisticated genetic systems available, largely as a consequence of their amenability to genetic manipulation via natural competence. Despite this, few options are available for the creation of markerless mutations in streptococci, especially within wild-type strains. Markerless mutagenesis is a critical tool for genetic studies, as it allows the user to explore many fundamental questions that are not easily addressable using marked mutagenesis. Here, we describe a new approach for streptococcal markerless mutagenesis that offers a variety of advantages over the current approach, which employs induced sensitivity to the toxic substrate 4-CP. The approach employed here should be readily adaptable for the creation of similar markerless mutagenesis systems in other organisms.

Toxin-Antitoxin (TA) systems are abundant in prokaryotes and play an important role in various biological processes such as plasmid maintenance, phage inhibition, stress response, biofilm formation, and dormant persister cell generation. TA loci are abundant in pathogenic intracellular micro-organisms and help in their adaptation to the harsh host environment such as nutrient deprivation, oxidation, immune response, and antimicrobials. Several studies have reported the involvement of TA loci in establishing successful infection, intracellular survival, better colonization, adaptation to host stresses, and chronic infection. Overall, the TA loci play a crucial role in bacterial virulence and pathogenesis. Nonetheless, there are some controversies about the role of TA system in stress response, biofilm and persister formation. In this review, we describe the role of the TA systems in bacterial virulence. We discuss the important features of each type of TA system and the recent discoveries identifying key contributions of TA loci in bacterial pathogenesis.

The chemical modification of cellular macromolecules by the transfer of ADP-ribose unit(s), known as ADP-ribosylation, is an ancient homeostatic and stress response control system. Highly conserved across the evolution, ADP-ribosyltransferases and ADP-ribosylhydrolases control ADP-ribosylation signalling and cellular responses. In addition to proteins, both prokaryotic and eukaryotic transferases can covalently link ADP-ribosylation to different conformations of nucleic acids, thus highlighting the evolutionary conservation of archaic stress response mechanisms. Here, we report several structural and functional aspects of DNA ADP-ribosylation modification controlled by the prototype DarT and DarG pair, which show ADP-ribosyltransferase and hydrolase activity, respectively. DarT/DarG is a toxin–antitoxin system conserved in many bacterial pathogens, for example in Mycobacterium tuberculosis, which regulates two clinically important processes for human health, namely, growth control and the anti-phage response. The chemical modulation of the DarT/DarG system by selective inhibitors may thus represent an exciting strategy to tackle resistance to current antimicrobial therapies. [ABSTRACT FROM AUTHOR]

Bacterial cell filamentation is a morphological change wherein cell division is blocked, which can improve bacterial survival under unfavorable conditions (e.g., antibiotic stress that causes DNA damage). As an extrachromosomal DNA molecule, plasmids can confer additionally advantageous traits including antibiotic resistance on the host. However, little is known about whether plasmids could shift bacterial morphological responses to antibiotic stress. Here, it is reported that plasmid‐free cells, rather than plasmid‐bearing cells, exhibit filamentation and asymmetrical cell division under exposure to sub‐inhibitory concentrations of antibiotics (ciprofloxacin and cephalexin). The underlying mechanism is revealed by investigating DNA damage, cell division inhibitor sulA, the SOS response, toxin‐antitoxin module (parDE) located on plasmids, and efflux pumps. Significantly higher expression of sulA is observed in plasmid‐free cells, compared to plasmid‐bearing cells. Plasmid carriage enables the hosts to suffer less DNA damage, exhibit stronger efflux pump activities, and thus have a higher antibiotic tolerance. These benefits are attributed to the parDE module that mediates stress responses from plasmid‐bearing cells and mainly contributes to cell morphological changes. Collectively, the findings demonstrate that plasmids can confer additional innate defenses on the host to antibiotics, thus advancing the understanding of how plasmids affect bacterial evolution in hostile environments. [ABSTRACT FROM AUTHOR]

Background: Curiosity on toxin–antitoxin modules has increased intensely over recent years as it is ubiquitously present in many bacterial genomes, including pathogens like Methicillin-resistant Staphylococcus aureus (MRSA). Several cellular functions of TA systems have been proposed however, their exact role in cellular physiology remains unresolved. Methods: This study aims to find out the impact of the mazEF toxin–antitoxin module on biofilm formation, pathogenesis, and antibiotic resistance in an isolated clinical ST239 MRSA strain, by constructing mazE and mazF mutants using CRISPR–cas9 base-editing plasmid (pnCasSA-BEC). Transcriptome analysis (RNA-seq) was performed for the mazE antitoxin mutant in order to identify the differentially regulated genes. The biofilm formation was also assessed for the mutant strains. Antibiogram profiling was carried out for both the generated mutants followed by murine experiment to determine the pathogenicity of the constructed strains. Results: For the first time our work showed, that MazF promotes cidA mediated cell death and lysis for biofilm formation without playing any significant role in host virulence as suggested by the murine experiment. Interestingly, the susceptibility to oxacillin, daptomycin and vancomycin was reduced significantly by the activated MazF toxin in the mazE mutant strain. Conclusions: Our study reveals that activated MazF toxin leads to resistance to antibiotics like oxacillin, daptomycin and vancomycin. Therefore, in the future, any potential antibacterial drug can be designed to target MazF toxin against the problematic multi-drug resistant bug. [ABSTRACT FROM AUTHOR]

Abstract Background Curiosity on toxin–antitoxin modules has increased intensely over recent years as it is ubiquitously present in many bacterial genomes, including pathogens like Methicillin-resistant Staphylococcus aureus (MRSA). Several cellular functions of TA systems have been proposed however, their exact role in cellular physiology remains unresolved. Methods This study aims to find out the impact of the mazEF toxin–antitoxin module on biofilm formation, pathogenesis, and antibiotic resistance in an isolated clinical ST239 MRSA strain, by constructing mazE and mazF mutants using CRISPR–cas9 base-editing plasmid (pnCasSA-BEC). Transcriptome analysis (RNA-seq) was performed for the mazE antitoxin mutant in order to identify the differentially regulated genes. The biofilm formation was also assessed for the mutant strains. Antibiogram profiling was carried out for both the generated mutants followed by murine experiment to determine the pathogenicity of the constructed strains. Results For the first time our work showed, that MazF promotes cidA mediated cell death and lysis for biofilm formation without playing any significant role in host virulence as suggested by the murine experiment. Interestingly, the susceptibility to oxacillin, daptomycin and vancomycin was reduced significantly by the activated MazF toxin in the mazE mutant strain. Conclusions Our study reveals that activated MazF toxin leads to resistance to antibiotics like oxacillin, daptomycin and vancomycin. Therefore, in the future, any potential antibacterial drug can be designed to target MazF toxin against the problematic multi-drug resistant bug.

Toxin-antitoxin (TA) systems are important for stress adaptation in prokaryotes, including persistence, antibiotic resistance, pathogenicity, and biofilm formation. Toxins can cause cell death, reversible growth stasis, and direct inhibition of crucial cellular processes through various mechanisms, while antitoxins neutralize the effects of toxins. In bacteria, these systems have been studied in detail, whereas their function in archaea remains elusive. During heat stress, the thermoacidophilic archaeon Sulfolobus acidocaldarius exhibited an increase in the expression of several bicistronic type II vapBC TA systems, with the highest expression observed in the vapBC4 system. In the current study, we performed a comprehensive biochemical characterization of the VapBC4 TA system, establishing it as a bonafide type II toxin-antitoxin system. The VapC4 toxin is shown to have high-temperature catalyzed RNase activity specific for mRNA and rRNA, while the VapB4 antitoxin inhibits the toxic activity of VapC4 by interacting with it. VapC4 toxin expression led to heat-induced persister-like cell formation, allowing the cell to cope with the stress. Furthermore, this study explored the impact of vapBC4 deletion on biofilm formation, whereby deletion of vapC4 led to increased biofilm formation, suggesting its role in regulating biofilm formation. Thus, during heat stress, the liberated VapC4 toxin in cells could potentially signal a preference for persister cell formation over biofilm growth. Thus, our findings shed light on the diverse roles of the VapC4 toxin in inhibiting translation, inducing persister cell formation, and regulating biofilm formation in S. acidocaldarius , enhancing our understanding of TA systems in archaea., Importance: This research enhances our knowledge of toxin-antitoxin (TA) systems in archaea, specifically in the thermoacidophilic archaeon Sulfolobus acidocaldarius . TA systems are widespread in both bacterial and archaeal genomes, indicating their evolutionary importance. However, their exact functions in archaeal cellular physiology are still not well understood. This study sheds light on the complex roles of TA systems and their critical involvement in archaeal stress adaptation, including persistence and biofilm formation. By focusing on S. acidocaldarius , which lives in habitats with fluctuating temperatures that can reach up to 90°C, the study reveals the unique challenges and survival mechanisms of this organism. The detailed biochemical analysis of the VapBC4 TA system, and its crucial role during heat stress, provides insights into how extremophiles can survive in harsh conditions. The findings of this study show the various functions of the VapC4 toxin, including inhibiting translation, inducing persister-like cell formation, and regulating biofilm formation. This knowledge improves our understanding of TA systems in thermoacidophiles and has broader implications for understanding how microorganisms adapt to extreme environments.

Toxin-antitoxin modules are present in many bacterial pathogens. The VapBC family is particularly abundant in members of the Mycobacterium tuberculosis complex, with 50 modules present in the M. tuberculosis genome. In type IIA modules, the VapB antitoxin protein binds to and inhibits the activity of the co-expressed cognate VapC toxin protein. VapB proteins may also bind to promoter region sequences and repress the expression of the vapB-vapC operon. Though VapB-VapC interactions can control the amount of free VapC toxin in the bacterial cell, the mechanisms that affect this interaction are poorly understood. Based on our recent finding of Ser/Thr phosphorylation of VapB proteins in M. tuberculosis , we substituted phosphomimetic or phosphoablative amino acids at the phosphorylation sites of two VapB proteins. We found that phosphomimetic substitution of VapB27 and VapB46 resulted in decreased interaction with their respective cognate VapC proteins, whereas phosphoablative substitution did not alter binding. Similarly, we determined that phosphomimetic substitution interfered with VapB binding to promoter region DNA sequences. Both decreased VapB-VapC interaction and decreased VapB repression of vapB-vapC operon transcription would result in increased free VapC in the M. tuberculosis cell. In growth inhibition experiments, M. tuberculosis strains expressing vapB46-vapC46 constructs containing a phosphoablative vapB mutation resulted in lower toxicity compared to a strain expressing native vapB46 , whereas similar or greater toxicity was observed in the strain expressing the phosphomimetic vapB mutation. These results identify a novel mechanism by which VapC toxicity activity can be regulated by VapB phosphorylation.IMPORTANCEIntracellular bacterial toxins are present in many bacterial pathogens and have been linked to bacterial survival in response to stresses encountered during infection. The activity of many toxins is regulated by a co-expressed antitoxin protein that binds to and sequesters the toxin protein. The mechanisms by which an antitoxin may respond to stresses to alter toxin activity are poorly understood. Here, we show that antitoxin interactions with its cognate toxin and with promoter DNA required for antitoxin and toxin expression can be altered by Ser/Thr phosphorylation of the antitoxin and, thus, affect toxin activity. This reversible modification may play an important role in regulating toxin activity within the bacterial cell in response to signals generated during infection., Competing Interests: The authors declare no conflict of interest.

Background and Objective: Shigella flexneri is a highly contagious Gram-negative bacterium that causes severe diarrhea, especially in children under ten years old. The biofilm formation in this species increases its resistance to antibiotics. Given the important role of toxin-antitoxin (TA) systems in the stability and survival of bacteria under stress condition, this study was aimed to evaluate the expression of genes encoding TA systems and Lon protease (lonp) as the main protein regulating the expression and function of these systems in this microorganism in terms of biofilm formation. Materials and Methods: In this study, the standard Shigella flexneri ATCC 12022 was used. After the bacteria culture on the specific culture medium, the ability to form biofilm was quantitatively evaluated by microtiter plate method. Then, the expression level of the mentioned genes was assessed compared to the control group using real-time PCR. Results: The results of microtiter plate test showed that the studied Shigella flexneri was a strong biofilm strain. The qPCR results showed an increase in gene expression of the studied toxins and Lon protease at 8 and 24 h following biofilm formation induction (P

Antimicrobial resistance (AMR) in bacteria is an important global health problem affecting humans, animals, and the environment. AMR is considered as one of the major components in the “global one health”. Misuse/overuse of antibiotics in any one of the segments can impact the integrity of the others. In the presence of antibiotic selective pressure, bacteria tend to develop several defense mechanisms, which include structural changes of the bacterial outer membrane, enzymatic processes, gene upregulation, mutations, adaptive resistance, and biofilm formation. Several components of mobile genetic elements (MGEs) play an important role in the dissemination of AMR. Each one of these components has a specific function that lasts long, irrespective of any antibiotic pressure. Integrative and conjugative elements (ICEs), insertion sequence elements (ISs), and transposons carry the antimicrobial resistance genes (ARGs) on different genetic backbones. Successful transfer of ARGs depends on the class of plasmids, regulons, ISs proximity, and type of recombination systems. Additionally, phage-bacterial networks play a major role in the transmission of ARGs, especially in bacteria from the environment and foods of animal origin. Several other functional attributes of bacteria also get successfully modified to acquire ARGs. These include efflux pumps, toxin-antitoxin systems, regulatory small RNAs, guanosine pentaphosphate signaling, quorum sensing, two-component system, and clustered regularly interspaced short palindromic repeats (CRISPR) systems. The metabolic and virulence state of bacteria is also associated with a range of genetic and phenotypic resistance mechanisms. In spite of the availability of a considerable information on AMR, the network associations between selection pressures and several of the components mentioned above are poorly understood. Understanding how a pathogen resists and regulates the ARGs in response to antimicrobials can help in controlling the development of resistance. Here, we provide an overview of the importance of genetic network and regulation of AMR in bacterial pathogens.

Toxin-antitoxin (TA) systems are widely present in bacterial genomes. They consist of stable toxins and unstable antitoxins that are classified into distinct groups based on their structure and biological activity. TA systems are mostly related to mobile genetic elements and can be easily acquired through horizontal gene transfer. The ubiquity of different homologous and non-homologous TA systems within a single bacterial genome raises questions about their potential cross-interactions. Unspecific cross-talk between toxins and antitoxins of non-cognate modules may unbalance the ratio of the interacting partners and cause an increase in the free toxin level, which can be deleterious to the cell. Moreover, TA systems can be involved in broadly understood molecular networks as transcriptional regulators of other genes’ expression or modulators of cellular mRNA stability. In nature, multiple copies of highly similar or identical TA systems are rather infrequent and probably represent a transition stage during evolution to complete insulation or decay of one of them. Nevertheless, several types of cross-interactions have been described in the literature to date. This implies a question of the possibility and consequences of the TA system cross-interactions, especially in the context of the practical application of the TA-based biotechnological and medical strategies, in which such TAs will be used outside their natural context, will be artificially introduced and induced in the new hosts. Thus, in this review, we discuss the prospective challenges of system cross-talks in the safety and effectiveness of TA system usage.

Bacteria use quorum sensing (QS) to communicate with each other via secreted small autoinducers produced by individuals. QS allows bacteria to display a unified response that benefits the species during adaptation to environment, colonization, and defense against competitors. In oral streptococci, the CSP-ComDE QS is an inducible DNA damage repair system that is pivotal for bacterial survival. In the oral pathogen Streptococcus mutans, the QS system positively influences the formation of antibiotic persisters, cells that can survive antibiotic attack by entering a non-proliferative state. We recently identified a novel gene, pep299, that is activated in the persister cell fraction induced by QS. In this study, we focused our investigation on the role of pep299, a gene encoding a bacteriocin-like peptide, in the formation of antibiotic persisters. Mutant Δ299, unable to produce Pep299, showed a dramatic reduction in the number of stress-induced persisters. Using a co-culture assay, we showed that cells overproducing pep299 induced the formation of persisters in the mutant, suggesting that Pep299 was actively secreted and detected by neighboring cells. Cells exposed to DNA damage conditions activated the gene expression of pep299. Interestingly, our results suggested that the pep299 gene was also involved in the regulation of a QS-inducible toxin–antitoxin system. Our study suggests that the pep299 gene is at the core of the triggered persistence phenotype in S. mutans, allowing cells to transition into a state of reduced metabolic activity and antibiotic tolerance. [ABSTRACT FROM AUTHOR]

ABSTRACT Toxin-antitoxin (TA) systems are genetic modules that consist of a stable protein-toxin and an unstable antitoxin that neutralizes the toxic effect. In type II TA systems, the antitoxin is a protein that inhibits the toxin by direct binding. Type II TA systems, whose roles and functions are under intensive study, are highly distributed among bacterial chromosomes. Here, we identified and characterized a novel type II TA system PrrT/A encoded in the chromosome of the clinical isolate 39016 of the opportunistic pathogen Pseudomonas aeruginosa. We have shown that the PrrT/A system exhibits classical type II TA characteristics and novel regulatory properties. Following deletion of the prrA antitoxin, we discovered that the system is involved in a range of processes including (i) biofilm and motility, (ii) reduced prophage induction and bacteriophage production, and (iii) increased fitness for aminoglycosides. Taken together, these results highlight the importance of this toxin-antitoxin system to key physiological traits in P. aeruginosa. IMPORTANCE The functions attributed to bacterial TA systems are controversial and remain largely unknown. Our study suggests new insights into the potential functions of bacterial TA systems. We reveal that a chromosome-encoded TA system can regulate biofilm and motility, antibiotic resistance, prophage gene expression, and phage production. The latter presents a thus far unreported function of bacterial TA systems. In addition, with the emergence of antimicrobial-resistant bacteria, especially with the rising of P. aeruginosa resistant strains, the investigation of TA systems is critical as it may account for potential new targets against the resistant strains.

Introduction: Toxin-antitoxin (TA) systems are widespread among bacteria, archaea and fungi. They are classified into six types (I-VI) and have recently been proposed as novel drug targets. Objectives: This study aimed to screen the pathogen Acinetobacter baumannii, known for its alarming antimicrobial resistance, for TA systems and identified a CptBA-like type IV TA, one of the least characterized systems. Methods: In silico methods included secondary structure prediction, comparative genomics, multiple sequence alignment, and phylogenetic analysis, while in vitro strategies included plasmid engineering and expression of the TA system in Escherichia coli BL21, growth measurement, and transcription analysis with quantitative reverse-transcription polymerase chain reaction. Results: Comparative genomics demonstrated the distribution of CptBA-like systems among Gram-negative bacteria, while phylogenetic analysis delineated two major groups, in each of which Acinetobacter spp. proteins clustered together. Sequence alignment indicated the conservation of cptA and cptB in 4,732 strains of A. baumannii in the same syntenic order. Using A. baumannii recombinant cptA and cptB, cloned under different promoters, confirmed their TA nature, as cptB expression was able to reverse growth inhibition by CptA in a dose-time dependent manner. Furthermore, transcriptional analysis of cptBA in clinical and standard A. baumannii strains demonstrated the downregulation of this system under oxidative and antibiotic stress. Conclusion: Combining in silico and in vitro studies confirmed the predicted TA nature of a cptBA-like system in A. baumannii . Transcriptional analysis suggests a possible role of cptBA in response to antibiotics and stress factors in A. baumannii, making it a promising drug target.

Toxin-antitoxins (TAs) are prokaryotic two-gene systems composed of a toxin neutralized by an antitoxin. Toxin-antitoxin-chaperone (TAC) systems additionally include a SecB-like chaperone that stabilizes the antitoxin by recognizing its chaperone addiction (ChAD) element. TACs mediate antiphage defense, but the mechanisms of viral sensing and restriction are unexplored. We identify two Escherichia coli antiphage TAC systems containing host inhibition of growth (HigBA) and CmdTA TA modules, HigBAC and CmdTAC. HigBAC is triggered through recognition of the gpV major tail protein of phage λ. Chaperone HigC recognizes gpV and ChAD via analogous aromatic molecular patterns, with gpV outcompeting ChAD to trigger toxicity. For CmdTAC, the CmdT ADP-ribosyltransferase toxin modifies mRNA to halt protein synthesis and limit phage propagation. Finally, we establish the modularity of TACs by creating a hybrid broad-spectrum antiphage system combining the CmdTA TA warhead with a HigC chaperone phage sensor. Collectively, these findings reveal the potential of TAC systems in broad-spectrum antiphage defense. [Display omitted] • E. coli HigBAC and CmdTAC are translation-targeting phage immunity TAC systems • The HigC chaperone senses the phage λ major tail protein to trigger the HigB toxin • The CmdT ADP-ribosyltransferase toxin inhibits translation by modifying mRNA • HigC combined with CmdTA yields a hybrid broad-spectrum antiphage defense system Toxin-antitoxin-chaperone (TAC) systems have been shown to mediate antiphage defense via as-yet unexplored mechanisms. Focusing on two TACs encoded by Escherichia coli prophages—HigBAC and CmdTAC—Mets et al. dissect the mechanisms of phage recognition by the SecB-like chaperone phage sensor and phage restriction by the TA warhead. [ABSTRACT FROM AUTHOR]

Composite genomic islands (GIs) are useful models for studying GI evolution if they can revert into the previous components. In this study, CGI48—a 48,135-bp native composite GI that carries GI21, whose homologies specifically integrated in the conserved yicC gene—were identified in Shewanella putrefaciens CN-32. CGI48 was integrated into the tRNATrp gene, which is a conserved gene locus for the integration of genomic islands in Shewanella. Upon expressing integrase and excisionase, CGI48 and GI21 are excised from chromosomes via site-specific recombination. The shorter attachment sites of GI21 facilitated the capture of GI21 into CGI48. Moreover, GI21 encodes a functional HipAB toxin–antitoxin system, thus contributing to the maintenance of CGI48 in the host bacteria. This study provides new insights into GI evolution by performing the excision process of the inserting GI and improves our understanding of the maintenance mechanisms of composite GI. [ABSTRACT FROM AUTHOR]

ABSTRACT Toxin-antitoxin systems are genetic elements that are widespread in prokaryotes. Although molecular mode of action of many of these toxins has been identified, their biological functions are mostly unknown. We investigated the functional integration of the TisB/IstR toxin-antitoxin system in the Escherichia coli SOS genotoxic stress response network. We showed that the tisB gene is induced in cells exposed to high doses of the genotoxic antibiotic trimethoprim. However, we also found that TisB contributes to trimethoprim-induced lethality. This is a consequence of the TisB-induced drop in the proton motive force (PMF), which results in blocking the thymine import and therefore the functioning of the pyrimidine salvage pathway. Conversely, a TisB-induced PMF drop protects cells by preventing the import of some other toxic compounds, like the aminoglycoside antibiotic gentamicin and colicin M, in the SOS-induced cells. Colicins are cytotoxic molecules produced by Enterobacterales when they are exposed to strong genotoxic stresses in order to compete with other microbiota members. We indeed found that TisB contributes to E. coli’s fitness during mouse gut colonization. Based on the results obtained here, we propose that the primary biological role of the TisB toxin is to increase the probability of survival and maintenance in the mammalian gut of their bacterial hosts when they have to simultaneously deal with massive DNA damages and a fierce chemical warfare with other microbiota members. IMPORTANCE The contribution of toxin-antitoxin systems to the persistence of bacteria to antibiotics has been intensively studied. This is also the case with the E. coli TisB/IstR toxin-antitoxin system, but the contribution of TisB to the persistence to antibiotics turned out to be not as straightforward as anticipated. In this study, we show that TisB can decrease, but also increase, cytotoxicity of different antibiotics. This inconsistency has a common origin, i.e., TisB-induced collapse of the PMF, which impacts the import and the action of different antibiotics. By taking into account the natural habitat of TisB bacterial hosts, the facts that this toxin-antitoxin system is integrated into the genotoxic stress response regulon SOS and that both SOS regulon and TisB are required for E. coli to colonize the host intestine, and the phenotypic consequences of the collapse of the PMF, we propose that TisB protects its hosts from cytotoxic molecules produced by competing intestinal bacteria.

Composite genomic islands (GIs) are useful models for studying GI evolution if they can revert into the previous components. In this study, CGI48—a 48,135-bp native composite GI that carries GI21, whose homologies specifically integrated in the conserved yicC gene—were identified in Shewanella putrefaciens CN-32. CGI48 was integrated into the tRNATrp gene, which is a conserved gene locus for the integration of genomic islands in Shewanella. Upon expressing integrase and excisionase, CGI48 and GI21 are excised from chromosomes via site-specific recombination. The shorter attachment sites of GI21 facilitated the capture of GI21 into CGI48. Moreover, GI21 encodes a functional HipAB toxin–antitoxin system, thus contributing to the maintenance of CGI48 in the host bacteria. This study provides new insights into GI evolution by performing the excision process of the inserting GI and improves our understanding of the maintenance mechanisms of composite GI.

Antimicrobial peptides (AMPs) are versatile molecules with broad antimicrobial activity produced by representatives of the three domains of life. Also, there are derivatives of AMPs and artificial short peptides that can inhibit microbial growth. Beyond killing microbes, AMPs at grow sub-inhibitory concentrations also exhibit antivirulence activity against critical pathogenic bacteria, including ESKAPE pathogens. Anti-virulence therapies are an alternative to antibiotics since they do not directly affect viability and growth, and they are considered less likely to generate resistance. Bacterial biofilms significantly increase antibiotic resistance and are linked to establishing chronic infections. Various AMPs can kill biofilm cells and eradicate infections in animal models. However, some can inhibit biofilm formation and promote dispersal at subgrowth inhibitory concentrations. These examples are discussed here, along with those of peptides that inhibit the expression of traits controlled by quorum sensing, such as the production of exoproteases, phenazines, surfactants, toxins, among others. In addition, specific targets that are determinants of virulence include secretion systems (type II, III, and VI) responsible for releasing effector proteins toxic to eukaryotic cells. This review summarizes the current knowledge on the anti-virulence properties of AMPs and the future directions of their research. [ABSTRACT FROM AUTHOR]

Abstract Background The origin of most of the Lactobacillus rhamnosus genome sequences lodged in NCBI can be traced to food and faecal isolates followed by blood and tissue sites but with minimal representation from oral and vaginal isolates. However, on the L. rhamnosus phylogenetic tree no apparent clade is linked to the origin of isolation or to the relevant clinical source, except for a distinct clade exclusively shared by L. rhamnosus isolates from early stages of dental pulp infection (LRHMDP2 and LRHMDP3) and from bronchoalveolar lavage (699_LRHA and 708_LRHA) from a critical care patient. These L. rhamnosus strains, LRHMDP2, LRHMDP3, 699_LRHA and 708_LRHA isolated from different continents, display closest genome neighbour gapped identity of 99.95%. The aim of this study was to define a potentially unique complement of genes of clinical relevance shared between these L. rhamnosus clinical isolates in comparison to probiotic L. rhamnosus strains. Results In this analysis we used orthologous protein identification tools such as ProteinOrtho followed by tblastn alignments to identify a novel tyrosine protein phosphatase (wzb)-tyrosine-protein kinase modulator EpsC (wzd)- synteny exopolysaccharide (EPS) cluster. This EPS cluster was specifically conserved in a clade of 5 clinical isolates containing the four L. rhamnosus clinical isolates noted above and Lactobacillus spp. HMSC077C11, a clinical isolate from a neck abscess. The EPS cluster was shared with only two other strains, L. rhamnosus BPL5 and BPL15, which formed a distant clade on the L. rhamnosus phylogenetic tree, with a closest genome neighbour gapped identity of 97.51% with L. rhamnosus LRHMDP2 and LRHMDP3. Exclusivity of this EPS cluster (from those identified before) was defined by five EPS genes, which were specifically conserved between the clade of 5 clinical isolates and L. rhamnosus BPL5 and BPL15 when compared to the remaining L. rhamnosus strains. Comparative genome analysis between the clade of 5 clinical isolates and L. rhamnosus BPL5 and BPL15 showed a set of 58 potentially unique genes characteristic of the clade of 5. Conclusion The potentially unique functional protein orthologs associated with the clade of 5 clinical isolates may provide understanding of fitness under selective pressure.

Background: Mycobacterium smegmatis and other nontuberculous mycobacteria (NTM) are widely distributed in the environment, but a significant increase of NTM infections has taken place in the last few decades. The objective of this study was to determine the role of toxin–antitoxin (TA) vapBC and mazEF systems that act as effectors of persistence in the stress response of NTM. Methods: The growth ability and the biofilm formation of NTM were evaluated by conventional methods. Bacterial cell viability was determined using MTT staining, agar plating, or the method of limiting dilutions. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of antibiotics were estimated using broth and agar dilution methods. Results: Despite a comparable growth dynamics and biofilm formation on solid/liquid interface with the wild type, a M. smegmatis vapBC, mazEF, and vapBC × mazEF deletion mutant produced more abundant pellicle and were more susceptible to heat shock. Significant differences were also found in the resistance wild type of NTM to isoniazid and ciprofloxacin reflected by higher MBC/MIC ratios. The proposed method of cultivation of agar blocks without visible growth after MIC determination into a liquid medium allows us to detect transition of all wild type of NTM strains to a dormant state in the presence of subMICs of isoniazid and ciprofloxacin while all deletion mutants failed to form dormant cells. Conclusion: Our data suggest that both vapBC and mazEF TA systems putatively involved in the heat and antibiotic stress response of NTM via their key role in transition to the dormant state.

Antimicrobial peptides (AMPs) are versatile molecules with broad antimicrobial activity produced by representatives of the three domains of life. Also, there are derivatives of AMPs and artificial short peptides that can inhibit microbial growth. Beyond killing microbes, AMPs at grow sub-inhibitory concentrations also exhibit anti-virulence activity against critical pathogenic bacteria, including ESKAPE pathogens. Anti-virulence therapies are an alternative to antibiotics since they do not directly affect viability and growth, and they are considered less likely to generate resistance. Bacterial biofilms significantly increase antibiotic resistance and are linked to establishing chronic infections. Various AMPs can kill biofilm cells and eradicate infections in animal models. However, some can inhibit biofilm formation and promote dispersal at sub-growth inhibitory concentrations. These examples are discussed here, along with those of peptides that inhibit the expression of traits controlled by quorum sensing, such as the production of exoproteases, phenazines, surfactants, toxins, among others. In addition, specific targets that are determinants of virulence include secretion systems (type II, III, and VI) responsible for releasing effector proteins toxic to eukaryotic cells. This review summarizes the current knowledge on the anti-virulence properties of AMPs and the future directions of their research.

The chemical modification of cellular macromolecules by the transfer of ADP-ribose unit(s), known as ADP-ribosylation, is an ancient homeostatic and stress response control system. Highly conserved across the evolution, ADP-ribosyltransferases and ADP-ribosylhydrolases control ADP-ribosylation signalling and cellular responses. In addition to proteins, both prokaryotic and eukaryotic transferases can covalently link ADP-ribosylation to different conformations of nucleic acids, thus highlighting the evolutionary conservation of archaic stress response mechanisms. Here, we report several structural and functional aspects of DNA ADP-ribosylation modification controlled by the prototype DarT and DarG pair, which show ADP-ribosyltransferase and hydrolase activity, respectively. DarT/DarG is a toxin–antitoxin system conserved in many bacterial pathogens, for example in Mycobacterium tuberculosis, which regulates two clinically important processes for human health, namely, growth control and the anti-phage response. The chemical modulation of the DarT/DarG system by selective inhibitors may thus represent an exciting strategy to tackle resistance to current antimicrobial therapies.

Burkholderia pseudomallei ( Bpm ) is a Gram-negative intracellular pathogen that causes melioidosis in humans, a neglected, underreported, and lethal disease that can reach a fatal outcome in over 50% of the cases. It can produce both acute and chronic infections, the latter being particularly challenging to eliminate because of the intracellular life cycle of the bacteria and its ability to generate a "persister" dormant state. The molecular mechanism that allows the switch between growing and persister phenotypes is not well understood but it is hypothesized to be due at least in part to the participation of toxin-antitoxin (TA) systems. We have previously studied the link between one of those systems (defined as HigBA) with specific expression patterns associated with levofloxacin antibiotic exposure. Through in silico methods, we predicted the presence of another three pairs of genes encoding for additional putative HigBA systems. Therefore, our main goal was to establish which mechanisms are conserved as well as which pathways are specific among different Bpm TA systems from the same family. We hypothesize that the high prevalence, and sometimes even redundancy of these systems in the Bpm chromosomes indicates that they can interact with each other and not function as only individual systems, as it was traditionally thought, and might be playing an undefined role in Bpm lifecycle. Here, we show that both the toxin and the antitoxin of the different systems contribute to bacterial survival and that toxins from the same family can have a cumulative effect under environmental stressful conditions., Importance: Toxin-antitoxin (TA) systems play a significant role in bacterial persistence, a phenomenon where bacterial cells enter a dormant or slow-growing state to survive adverse conditions such as nutrient deprivation, antibiotic exposure, or host immune responses. By studying TA systems in Burkholderia pseudomallei , we can gain insights into how this pathogen survives and persists in the host environment, contributing to its virulence and ability to cause melioidosis chronic infections., Competing Interests: The authors declare no conflict of interest.

Bacterial chromosomal type I toxin-antitoxin systems consist of a small protein, typically under 60 amino acids, and a small RNA (sRNA) that represses toxin translation. These gene pairs have gained attention over the last decade for their contribution to antibiotic persistence and phage tolerance in bacteria. However, biological functions for many remain elusive as gene deletions often fail to produce an observable phenotype. For many pairs, it is still unknown when the toxin and/or antitoxin gene are natively expressed within the bacterium. We examined sequence conservation of three type I toxin-antitoxin systems, tisB/istR-1, shoB/ohsC, and zor/orz , in over 2,000 Escherichia coli strains, including pathogenic and commensal isolates. Using our custom database, we found that these gene pairs are widespread across E. coli and have expression potential via BLASTn. We identified an alternative, dominant sequence variant of TisB and confirmed that it is toxic upon overproduction. Additionally, analyses revealed a highly conserved sequence in the zorO mRNA untranslated region that is required for full toxicity. We further noted that over 30% of E. coli genomes contain an orz antitoxin gene only and confirmed its expression in a representative strain: the first confirmed report of a type I antitoxin without its cognate toxin. Our results add to our understanding of these systems, and our methodology is applicable for other type I loci to identify critical regulatory and functional features.IMPORTANCEChromosomal type I toxin-antitoxins are a class of genes that have gained increasing attention over the last decade for their roles in antibiotic persistence which may contribute to therapeutic failures. However, the control of many of these genes and when they function have remained elusive. We demonstrate that a simple genetic conservation-based approach utilizing free, publicly available data yields known and novel insights into the regulation and function of three chromosomal type I toxin-antitoxins in Escherichia coli . This study also provides a framework for how this approach could be applied to other genes of interest., Competing Interests: The authors declare no conflict of interest.

Noncoding RNAs (ncRNA) have emerged as important components of regulatory networks governing bacterial physiology and virulence. Previous deep-sequencing analysis identified a large diversity of ncRNAs in the human enteropathogen Clostridioides (Clostridium) difficile. Some of them are trans-encoded RNAs that could require the RNA chaperone protein Hfq for their action. Recent analysis suggested a pleiotropic role of Hfq in C. difficile with the most pronounced effect on sporulation, a key process during the infectious cycle of this pathogen. However, a global view of RNAs interacting with C. difficile Hfq is missing. In the present study, we performed RNA immunoprecipitation high-throughput sequencing (RIP-Seq) to identify Hfq-associated RNAs in C. difficile. Our work revealed a large set of Hfq-interacting mRNAs and ncRNAs, including mRNA leaders and coding regions, known and potential new ncRNAs. In addition to trans-encoded RNAs, new categories of Hfq ligands were found including cis-antisense RNAs, riboswitches and CRISPR RNAs. ncRNA-mRNA and ncRNA-ncRNA pairings were postulated through computational predictions. Investigation of one of the Hfq-associated ncRNAs, RCd1, suggests that this RNA contributes to the control of late stages of sporulation in C. difficile. Altogether, these data provide essential molecular basis for further studies of post-transcriptional regulatory network in this enteropathogen. [ABSTRACT FROM AUTHOR]

Background and Aim: Shigella flexneri is a highly contagious Gram-negative bacterium that causes severe diarrhea, especially in children under ten years old. The biofilm formation in this species increases its resistance to antibiotics. Given the important role of toxin-antitoxin (TA) systems in the stability and survival of bacteria under stress condition, this study was aimed to evaluate the expression of genes encoding TA systems and Lon protease (lonp) as the main protein regulating the expression and function of these systems in this microorganism in terms of biofilm formation. Materials and Methods: In this study, the standard Shigella f. ATCC 12022 was used. After the bacteria culture on the specific culture medium, the ability to form biofilm was quantitatively evaluated by microtiter plate method. Then, the expression level of the mentioned genes was assessed compared to the control group using real-time PCR. Results: The results of microtiter plate test showed that the studied Shigella f. was a strong biofilm strain. The qPCR results showed an increase in gene expression of the studied toxins and Lon protease at 8 and 24 h following biofilm formation induction (P<0.01). Conclusion: Due to the significant increase in the expression of the studied genes, especially Lon protease, GNAT and maz toxins at 24 h after biofilm formation, they can be potentially used as antimicrobial targets in new studies. [ABSTRACT FROM AUTHOR]

ABSTRACT Bacterial toxin-antitoxin (TA) systems encode a toxin and an antitoxin that counteracts the toxin. Such TA systems are found abundantly on bacterial chromosomes and on extrachromosomal genetic elements. The toxin is always a protein. Based on the nature of the antitoxin (protein or RNA) and on their mode of regulation, they are classified into six groups (I to VI). In the group II TA systems, both the toxin and the antitoxin are proteins, and the gene specifying the antitoxin precedes the gene specifying for the toxin. Here, we studied the intracellular localization in Escherichia coli cells of the proteins specified by the following type II TA modules: mazEF, chpBIK, mqsRA, and rnlAB. We visualized the localization of these proteins by fusing them with the fluorescent protein mCherry using recombinant DNA technology. We used fluorescence microscopy and image analysis software to obtain and quantify protein distribution data. With the exception of the chpBIK TA module, we found that the localization of each toxin-antitoxin complex was different from the localization of the toxin itself. Our results demonstrate clearly that the presence of the antitoxin shifts the localization of its respective toxin toward the middle of the cell, which could contribute to the reduction of cellular toxicity. IMPORTANCE Bacterial toxin-antitoxin (TA) systems, which were discovered in 1985, have since been studied extensively. These studies have focused particularly on the distribution of these bacterial TA systems on either plasmids or on bacterial chromosomes, their functionality, their targets, their relation to virulence, and their mechanisms of action. Our study, reported here, is the first to clarify the intracellular localization of the proteins specified for some type II TA systems. We have shown that, with the exception of the chpBIK module, each toxin-antitoxin complex was localized in a different part of the cell than the toxin itself. Our results revealed clearly that the presence of the antitoxin changes the localization of the toxin by moving the toxin toward the middle of the cell. Until now, the general view has been that the antagonistic effect of the antitoxins over their cognate toxins is based only on their direct structural interactions. Here, we show that this antagonistic effect is also a function of a specific change in the intracellular localization of the toxin.

The Type VI Secretion System (T6SS) has important roles relating to bacterial antagonism, subversion of host cells, and niche colonisation. Campylobacter jejuni is one of the leading bacterial causes of human gastroenteritis worldwide and is a commensal coloniser of birds. Although recently discovered, the T6SS biological functions and identities of its effectors are still poorly defined in C. jejuni. Here, we perform a comprehensive bioinformatic analysis of the C. jejuni T6SS by investigating the prevalence and genetic architecture of the T6SS in 513 publicly available genomes using C. jejuni 488 strain as reference. A unique and conserved T6SS cluster associated with the Campylobacter jejuni Integrated Element 3 (CJIE3) was identified in the genomes of 117 strains. Analyses of the T6SS-positive 488 strain against the T6SS-negative C. jejuni RM1221 strain and the T6SS-positive plasmid pCJDM202 carried by C. jejuni WP2-202 strain defined the “T6SS-containing CJIE3” as a pathogenicity island, thus renamed as Campylobacter jejuni Pathogenicity Island-1 (CJPI-1). Analysis of CJPI-1 revealed two canonical VgrG homologues, CJ488_0978 and CJ488_0998, harbouring distinct C-termini in a genetically variable region downstream of the T6SS operon. CJPI-1 was also found to carry a putative DinJ-YafQ Type II toxin-antitoxin (TA) module, conserved across pCJDM202 and the genomic island CJIE3, as well as several open reading frames functionally predicted to encode for nucleases, lipases, and peptidoglycan hydrolases. This comprehensive in silico study provides a framework for experimental characterisation of T6SS-related effectors and TA modules in C. jejuni.