Escherichia coli MazF Leads to the Simultaneous Selective Synthesis of Both “Death Proteins” and “Survival Proteins”

The Escherichia coli mazEF module is one of the most thoroughly studied toxin–antitoxin systems. mazF encodes a stable toxin, MazF, and mazE encodes a labile antitoxin, MazE, which prevents the lethal effect of MazF. MazF is an endoribonuclease that leads to the inhibition of protein synthesis by cleaving mRNAs at ACA sequences. Here, using 2D-gels, we show that in E. coli, although MazF induction leads to the inhibition of the synthesis of most proteins, the synthesis of an exclusive group of proteins, mostly smaller than about 20 kDa, is still permitted. We identified some of those small proteins by mass spectrometry. By deleting the genes encoding those proteins from the E. coli chromosome, we showed that they were required for the death of most of the cellular population. Under the same experimental conditions, which induce mazEF-mediated cell death, other such proteins were found to be required for the survival of a small sub-population of cells. Thus, MazF appears to be a regulator that induces downstream pathways leading to death of most of the population and the continued survival of a small sub-population, which will likely become the nucleus of a new population when growth conditions become less stressful

A Differential Effect of E. coli Toxin-Antitoxin Systems on Cell Death in Liquid Media and Biofilm Formation

Toxin-antitoxin (TA) modules are gene pairs specifying for a toxin and its antitoxin and are found on the chromosomes of many bacteria including pathogens. Here we report how each of five such TA systems in E. coli affect bacterial cell death differently in liquid media and during biofilm formation. Of all these systems, only the TA system mazEF mediated cell death both in liquid media and during biofilm formation. At the other extreme, as our results have revealed here, the TA system dinJ-YafQ is unique in that it is involved only in the death process during biofilm formation. Cell death governed by mazEF and dinJ-YafQ seems to participate in biofilm formation through a novel mechanism

Removal of Hepatitis C Virus-Infected Cells by a Zymogenized Bacterial Toxin

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and has become a global health threat. No HCV vaccine is currently available and treatment with antiviral therapy is associated with adverse side effects. Moreover, there is no preventive therapy for recurrent hepatitis C post liver transplantation. The NS3 serine protease is necessary for HCV replication and represents a prime target for developing anti HCV therapies. Recently we described a therapeutic approach for eradication of HCV infected cells that is based on protein delivery of two NS3 protease-activatable recombinant toxins we named “zymoxins”. These toxins were inactivated by fusion to rationally designed inhibitory peptides via NS3-cleavable linkers. Once delivered to cells where NS3 protease is present, the inhibitory peptide is removed resulting in re-activation of cytotoxic activity. The zymoxins we described suffered from two limitations: they required high levels of protease for activation and had basal activities in the un-activated form that resulted in a narrow potential therapeutic window. Here, we present a solution that overcame the major limitations of the “first generation zymoxins” by converting MazF ribonuclease, the toxic component of the E. coli chromosomal MazEF toxin-antitoxin system, into an NS3-activated zymoxin that is introduced to cells by means of gene delivery. We constructed an expression cassette that encodes for a single polypeptide that incorporates both the toxin and a fragment of its potent natural antidote, MazE, linked via an NS3-cleavable linker. While covalently paired to its inhibitor, the ribonuclease is well tolerated when expressed in naïve, healthy cells. In contrast, activating proteolysis that is induced by even low levels of NS3, results in an eradication of NS3 expressing model cells and HCV infected cells. Zymoxins may thus become a valuable tool in eradicating cells infected by intracellular pathogens that express intracellular proteases

Characterization of the Interaction and Cross-Regulation of Three Mycobacterium tuberculosis RelBE Modules

RelBE represents a typical bacterial toxin-antitoxin (TA) system. Mycobacterium tuberculosis H37Rv, the pathogen responsible for human tuberculosis, contains three RelBE-like modules, RelBE, RelFG, and RelJK, which are at least partly expressed in human macrophages during infection. RelBE modules appear to be autoregulated in an atypical manner compared to other TA systems; however, the molecular mechanisms and potential interactions between different RelBE modules remain to be elucidated. In the present study, we characterized the interaction and cross-regulation of these Rel toxin-antitoxin modules from this unique pathogen. The physical interactions between the three pairs of RelBE proteins were confirmed and the DNA-binding domain recognized by three RelBE-like pairs and domain structure characteristics were described. The three RelE-like proteins physically interacted with the same RelB-like protein, and could conditionally regulate its binding with promoter DNA. The RelBE-like modules exerted complex cross-regulation effects on mycobacterial growth. The relB antitoxin gene could replace relF in cross-neutralizing the relG toxin gene. Conversely, relF enhanced the toxicity of the relE toxin gene, while relB increased the toxicity of relK. This is the first report of interactions between different pairs of RelBE modules of M. tuberculosis

PuLSE:Quality control and quantification of peptide sequences explored by phage display libraries

The design of highly diverse phage display libraries is based on assumption that DNA bases are incorporated at similar rates within the randomized sequence. As library complexity increases and expected copy numbers of unique sequences decrease, the exploration of library space becomes sparser and the presence of truly random sequences becomes critical. We present the program PuLSE (Phage Library Sequence Evaluation) as a tool for assessing randomness and therefore diversity of phage display libraries. PuLSE runs on a collection of sequence reads in the fastq file format and generates tables profiling the library in terms of unique DNA sequence counts and positions, translated peptide sequences, and normalized 'expected' occurrences from base to residue codon frequencies. The output allows at-a-glance quantitative quality control of a phage library in terms of sequence coverage both at the DNA base and translated protein residue level, which has been missing from toolsets and literature. The open source program PuLSE is available in two formats, a C++ source code package for compilation and integration into existing bioinformatics pipelines and precompiled binaries for ease of use

Role of RecA and the SOS Response in Thymineless Death in Escherichia coli

Thymineless death (TLD) is a classic and enigmatic phenomenon, documented in bacterial, yeast, and human cells, whereby cells lose viability rapidly when deprived of thymine. Despite its being the essential mode of action of important chemotherapeutic agents, and despite having been studied extensively for decades, the basic mechanisms of TLD have remained elusive. In Escherichia coli, several proteins involved in homologous recombination (HR) are required for TLD, however, surprisingly, RecA, the central HR protein and activator of the SOS DNA–damage response was reported not to be. We demonstrate that RecA and the SOS response are required for a substantial fraction of TLD. We show that some of the Rec proteins implicated previously promote TLD via facilitating activation of the SOS response and that, of the roughly 40 proteins upregulated by SOS, SulA, an SOS–inducible inhibitor of cell division, accounts for most or all of how SOS causes TLD. The data imply that much of TLD results from an irreversible cell-cycle checkpoint due to blocked cell division. FISH analyses of the DNA in cells undergoing TLD reveal blocked replication and apparent DNA loss with the region near the replication origin underrepresented initially and the region near the terminus lost later. Models implicating formation of single-strand DNA at blocked replication forks, a SulA-blocked cell cycle, and RecQ/RecJ-catalyzed DNA degradation and HR are discussed. The data predict the importance of DNA damage-response and HR networks to TLD and chemotherapy resistance in humans

Rapid and Sensitive Detection of an Intracellular Pathogen in Human Peripheral Leukocytes with Hybridizing Magnetic Relaxation Nanosensors

Bacterial infections are still a major global healthcare problem. The quick and sensitive detection of pathogens responsible for these infections would facilitate correct diagnosis of the disease and expedite treatment. Of major importance are intracellular slow-growing pathogens that reside within peripheral leukocytes, evading recognition by the immune system and detection by traditional culture methods. Herein, we report the use of hybridizing magnetic nanosensors (hMRS) for the detection of an intracellular pathogen, Mycobacterium avium spp. paratuberculosis (MAP). The hMRS are designed to bind to a unique genomic sequence found in the MAP genome, causing significant changes in the sample’s magnetic resonance signal. Clinically relevant samples, including tissue and blood, were screened with hMRS and results were compared with traditional PCR analysis. Within less than an hour, the hMRS identified MAP-positive samples in a library of laboratory cultures, clinical isolates, blood and homogenized tissues. Comparison of the hMRS with culture methods in terms of prediction of disease state revealed that the hMRS outperformed established culture methods, while being significantly faster (1 hour vs 12 weeks). Additionally, using a single instrument and one nanoparticle preparation we were able to detect the intracellular bacterial target in clinical samples at the genomic and epitope levels. Overall, since the nanoparticles are robust in diverse environmental settings and substantially more affordable than PCR enzymes, the potential clinical and field-based use of hMRS in the multiplexed identification of microbial pathogens and other disease-related biomarkers via a single, deployable instrument in clinical and complex environmental samples is foreseen

Evidence of Transfer by Conjugation of Type IV Secretion System Genes between Bartonella Species and Rhizobium radiobacter in Amoeba

Background: Bartonella species cospeciate with mammals and live within erythrocytes. Even in these specific niches, it has been recently suggested by bioinformatic analysis of full genome sequences that Lateral Gene Transfer (LGT) may occur but this has never been demonstrated biologically. Here we describe the sequence of the B. rattaustraliani (AUST/NH4 T) circular plasmid (pNH4) that encodes the tra cluster of the Type IV secretion system (T4SS) and we eventually provide evidence that Bartonella species may conjugate and exchange this plasmid inside amoeba. Principal Findings: The T4SS of pNH4 is critical for intracellular viability of bacterial pathogens, exhibits bioinformatic evidence of LGT among bacteria living in phagocytic protists. For instance, 3 out of 4 T4SS encoding genes from pNH4 appear to be closely related to Rhizobiales, suggesting that gene exchange occurs between intracellular bacteria from mammals (bartonellae) and plants (Rhizobiales). We show that B. rattaustraliani and Rhizobium radiobacter both survived within the amoeba Acanthamoeba polyphaga and can conjugate together. Our findings further support the hypothesis that tra genes might also move into and out of bacterial communities by conjugation, which might be the primary means of genomic evolution for intracellular adaptation by cross-talk of interchangeable genes between Bartonella species and plant pathogens. Conclusions: Based on this, we speculate that amoeba favor the transfer of genes as phagocytic protists, which allows fo

A Toxin-Antitoxin Module in Bacillus subtilis Can Both Mitigate and Amplify Effects of Lethal Stress

Bacterial type-2 (protein-protein) toxin-antitoxin (TA) modules are two-gene operons that are thought to participate in the response to stress. Previous work with Escherichia coli has led to a debate in which some investigators conclude that the modules protect from stress, while others argue that they amplify lethal stress and lead to programmed cell death. To avoid ambiguity arising from the presence of multiple TA modules in E. coli, the effect of the sole type-2 toxin-antitoxin module of Bacillus subtilis was examined for several types of lethal stress.Genetic knockout of the toxin gene, ndoA (ydcE), conferred protection to lethal stressors that included kanamycin, moxifloxacin, hydrogen peroxide, and UV irradiation. However, at low doses of UV irradiation the ndoA deficiency increased lethality. Indeed, gradually increasing UV dose with the ndoA mutant revealed a crossover response--from the mutant being more sensitive than wild-type cells to being less sensitive. For high temperature and nutrient starvation, the toxin deficiency rendered cells hypersensitive. The ndoA deficiency also reduced sporulation frequency, indicating a role for toxin-antitoxin modules in this developmental process. In the case of lethal antimicrobial treatment, deletion of the toxin eliminated a surge in hydrogen peroxide accumulation observed in wild-type cells.A single toxin-antitoxin module can mediate two opposing effects of stress, one that lowers lethality and another that raises it. Protective effects are thought to arise from toxin-mediated inhibition of translation based on published work. The enhanced, stress-mediated killing probably involves toxin-dependent accumulation of reactive oxygen species, since a deficiency in the NdoA toxin suppressed peroxide accumulation following antimicrobial treatment. The type and perhaps the level of stress appear to be important for determining whether this toxin will have a protective or detrimental effect

Plasmids and Rickettsial Evolution: Insight from Rickettsia felis

BACKGROUND: The genome sequence of Rickettsia felis revealed a number of rickettsial genetic anomalies that likely contribute not only to a large genome size relative to other rickettsiae, but also to phenotypic oddities that have confounded the categorization of R. felis as either typhus group (TG) or spotted fever group (SFG) rickettsiae. Most intriguing was the first report from rickettsiae of a conjugative plasmid (pRF) that contains 68 putative open reading frames, several of which are predicted to encode proteins with high similarity to conjugative machinery in other plasmid-containing bacteria. METHODOLOGY/PRINCIPAL FINDINGS: Using phylogeny estimation, we determined the mode of inheritance of pRF genes relative to conserved rickettsial chromosomal genes. Phylogenies of chromosomal genes were in agreement with other published rickettsial trees. However, phylogenies including pRF genes yielded different topologies and suggest a close relationship between pRF and ancestral group (AG) rickettsiae, including the recently completed genome of R. bellii str. RML369-C. This relatedness is further supported by the distribution of pRF genes across other rickettsiae, as 10 pRF genes (or inactive derivatives) also occur in AG (but not SFG) rickettsiae, with five of these genes characteristic of typical plasmids. Detailed characterization of pRF genes resulted in two novel findings: the identification of oriV and replication termination regions, and the likelihood that a second proposed plasmid, pRFδ, is an artifact of the original genome assembly. CONCLUSION/SIGNIFICANCE: Altogether, we propose a new rickettsial classification scheme with the addition of a fourth lineage, transitional group (TRG) rickettsiae, that is unique from TG and SFG rickettsiae and harbors genes from possible exchanges with AG rickettsiae via conjugation. We offer insight into the evolution of a plastic plasmid system in rickettsiae, including the role plasmids may have played in the acquirement of virulence traits in pathogenic strains, and the likely origin of plasmids within the rickettsial tree