Biology subjects

Lazinski, D. W.

Publications and source records attributed to Lazinski, D. W..

7 recordsLinked to original sources

bioRxiv2026

Kappa Prophage Impacts Vibrio cholerae Fitness and is Restricted by BREX Phage Defense System

Vibrio cholerae's pathogenicity largely stems from the acquisition of cholera toxin genes via lysogenic conversion mediated by the CTX prophage. However, the strong association of other prophages with epidemic strains has not been linked to lysogenic conversion. During the first wave of the seventh cholera pandemic, the Kappa prophage was commonly found in clinical isolates and was later shown to confer a competitive fitness advantage in the environment and to accelerate evolution by stimulating horizontal gene transfer; however, its role in virulence remains unknown. Here, we report that the Kappa prophage confers a competitive fitness advantage in vitro and during intestinal tract infection in both Wave 1 and Wave 3 strains. This competitive fitness advantage is due to phage predation on neighboring cells, and not to lysogenic conversion. Furthermore, we show that loss of Kappa coincides with the acquisition of novel phage defense systems from SXT ICEs VchBan9 and VchInd5. Phage defense systems BREX and AbiC inhibited Kappa predation and restricted horizontal gene transfer, thereby reducing Kappas competitive fitness advantage.

microbiology↗

bioRxiv2024

A Vibrio cholerae Anti-Phage System Depletes Nicotinamide Adenine Dinucleotide to Restrict Virulent Bacteriophages

Bacteria and their predatory viruses (bacteriophages or phages) are in a perpetual molecular arms race. This has led to the evolution of numerous phage defensive systems in bacteria that are still being discovered, as well as numerous ways of interference or circumvention on the part of phages. Here, we identify a unique molecular battle between the classical biotype of Vibrio cholerae and virulent phages ICP1, ICP2, and ICP3. We show that classical biotype strains resist almost all isolates of these phages due to a 25-kb genomic island harboring several putative anti-phage systems. We observed that one of these systems, Nezha, encoding SIR2-like and helicase proteins, inhibited the replication of all three phages. Bacterial SIR2-like enzymes degrade the essential metabolic coenzyme nicotinamide adenine dinucleotide (NAD+), thereby preventing replication of the invading phage. In support of this mechanism, we identified one phage isolate, ICP1_2001, which circumvents Nezha by encoding two putative NAD+ regeneration enzymes. By restoring the NAD+ pool, we hypothesize that this system antagonizes Nezha without directly interacting with either protein and should be able to antagonize other anti-phage systems that deplete NAD+.

microbiology↗

bioRxiv2021

A genetic interaction screen in Streptococcus pneumoniae identifies functionally redundant vaccine candidate proteins CbpC and CbpJ

Streptococcus pneumoniae is a Gram-positive bacterium that asymptomatically colonizes the nasopharynx and can disseminate to sterile sites resulting in pneumococcal diseases such as pneumonia, otitis media, bacteremia, and meningitis. Due to increased incidence of invasive disease caused by serotypes that are not included in available polysaccharide vaccines, there is a need for a broadly protective protein vaccine to complement the polysaccharide based vaccines. To limit immune escape such a vaccine would ideally target proteins that are essential for virulence. However, the genetic robustness of S. pneumoniae results in few surface exposed proteins being essential for virulence. Here we carried out a genetic interaction screen to identify functionally redundant surface protein pairs that could be used as bivalent protein vaccines, based on the observation that together, these protein pairs are essential for virulence. We identified four pairs of functionally redundant surface proteins that displayed a significant competitive disadvantage during murine pneumococcal pneumonia. Immunization with the most attenuated pair, CbpC and CbpJ, resulted in production of high titers of specific antibodies and a modest increased median survival times of mice challenged with pneumococcal pneumonia. This study demonstrates a method to identify essential pairs of surface-associated virulence proteins that could be widely applied to many bacterial pathogens. IMPORTANCEInfection by Streptococcus pneumoniae can result in life-threatening illness. Current licensed polysaccharide vaccines only protect against serotypes that are present in the vaccine - at most 23 serotypes of the total 100 identified, circulating serotypes. There remains a need for a widely protective protein vaccine that is effective against most circulating strains of S. pneumoniae. The significance of our research is in developing a method to identify functionally redundant protein pairs as potential vaccine candidates, which could inform the development of effective bivalent protein vaccines for many bacterial pathogens.

microbiology↗

bioRxiv2021

A Tail Fiber Protein and a Receptor-Binding Protein Mediate ICP2 Bacteriophage Interactions with Vibrio cholerae OmpU

ICP2 is a virulent bacteriophage (phage) that preys on Vibrio cholerae. ICP2 was first isolated from cholera patient stool samples. Some of these stools also contained ICP2-resistant isogenic V. cholerae strains harboring missense mutations in the trimeric outer membrane porin protein OmpU, identifying it as the ICP2 receptor. In this study, we identify the ICP2 proteins that mediate interactions with OmpU by selecting for ICP2 host-range mutants within infant rabbits infected with a mixture of wild type and OmpU mutant strains. ICP2 host-range mutants had missense mutations in putative tail fiber gene gp25 and putative adhesin gp23. Using site-specific mutagenesis we show that single or double mutations in gp25 are sufficient to generate the host-range mutant phenotype. However, at least one additional mutation in gp23 is required for robust plaque formation on specific OmpU mutants. Mutations in gp23 alone were insufficient to give a host-range mutant phenotype. All ICP2 host-range mutants retained the ability to plaque on wild type V. cholerae cells. The strength of binding of host-range mutants to V. cholerae correlated with plaque morphology, indicating that the selected mutations in gp25 and gp23 restore molecular interactions with the receptor. We propose that ICP2 host-range mutants evolve by a two-step process where, first, gp25 mutations are selected for their broad host-range, albeit accompanied by low level phage adsorption. Subsequent selection occurs for gp23 mutations that further increase productive binding to specific OmpU alleles, allowing for near wild type efficiencies of adsorption and subsequent phage multiplication. ImportanceConcern over multidrug-resistant bacterial pathogens, including Vibrio cholerae, has led to a renewed interest in phage biology and their potential for phage therapy. ICP2 is a genetically unique virulent phage isolated from cholera patient stool samples. It is also one of three phages in a prophylactic cocktail shown to be effective in animal models of infection and the only one of the three that requires a protein receptor (OmpU). This study identifies a ICP2 tail fiber and a receptor binding protein and examines how ICP2 responds to the selective pressures of phage-resistant OmpU mutants. We found that this particular co-evolutionary arms race presents fitness costs to both ICP2 and V. cholerae.

microbiology↗

bioRxiv2020

Identification of spacer and protospacer sequence requirements in the Vibrio cholerae Type I-E CRISPR/Cas System

The prokaryotic adaptive immune system CRISPR/Cas serves as defense against bacteriophage and invasive nucleic acid. A Type I-E CRISPR/Cas system has been detected in classical biotype isolates of Vibrio cholerae, the causative agent of the disease cholera. Experimental characterization of this system revealed a functional immune system that operates using a 5-TT-3 protospacer-adjacent motif (PAM) for interference. However, several designed spacers against the 5-TT-3 PAM do not interfere as expected, indicating further investigation of this system is necessary. In this study, we identified additional sequence requirements of a pyrimidine in the 5 position of the spacer and purine in the complementary position of the protospacer using 873 unique spacers and 2267 protospacers mined from CRISPR arrays in deposited sequences of V. cholerae. We present bioinformatic evidence that during acquisition the protospacer purine is captured in the prespacer and that a 5-RTT-3 PAM is necessary for spacer acquisition. Finally, we demonstrate experimentally that a 5-RTT-3 PAM is necessary for CRISPR interference by designing and manipulating spacer and cognate PAMs in a plasmid conjugation assay and discover functional consequences of base pairing with the 5 spacer pyrimidine in spacer efficacy. ImportanceBacterial CRISPR/Cas systems provide immunity by defending against phage and other invading elements. A thorough comprehension of the molecular mechanisms employed by these diverse systems will improve our understanding of bacteriophage-bacterial interactions and bacterial adaptation to foreign DNA. The Vibrio cholerae Type I-E system was previously identified in an extinct classical biotype and was partially characterized for its function. Here, using both bioinformatic and functional assays, we extend that initial study. We have found that the Type I-E system still exists in modern strains of V. cholerae. Furthermore, we defined additional sequence elements in both the CRISPR array and in target DNA that are required for immunity. CRISPR/Cas systems are now commonly used as precise and powerful genetic engineering tools. Knowledge of the sequences required for CRISPR/Cas immunity is a prerequisite for the effective design and experimental use of these systems. Our results greatly facilitate the effective use of one such system. Furthermore, we provide a publicly available script that assists in the detection and validation of CRISPR/Cas immunity requirements when such a system exists in any bacterial species.

microbiology↗

bioRxiv2020

Antibiotic hypersensitivity signatures identify targets for attack in the Acinetobacter baumannii cell envelope

Acinetobacter baumannii is an opportunistic pathogen that is a critical, high-priority target for new antibiotic development. Clearing of A. baumannii requires relatively high doses of antibiotics across the spectrum, primarily due to its protective cell envelope. Many of the proteins that support envelope integrity and modulate drug action are uncharacterized, largely because there is an absence of orthologs for several proteins that perform essential envelope-associated processes, impeding progress on this front. To identify targets that can synergize with current antibiotics, we performed an exhaustive analysis of A. baumannii mutants causing hypersensitivity to a multitude of antibiotic treatments. By examining mutants with antibiotic hypersensitivity profiles that parallel mutations in proteins of known function, we show that the function of poorly annotated proteins can be predicted and used to identify candidate missing link proteins in essential A. baumannii processes. Using this strategy, we uncovered multiple uncharacterized proteins with critical roles in cell division or cell elongation, and revealed that a predicted cell wall D,D-endopeptidase has an unappreciated function in lipooligosaccharide synthesis. Moreover, we provide a genetic strategy that uses hypersensitivity signatures to predict drug synergies, allowing the identification of {beta}-lactams that work cooperatively based on the cell wall assembly machineries that they preferentially target. These data reveal multiple pathways critical for envelope growth in A. baumannii that can be targeted in combination strategies for attacking the pathogen.

microbiology↗

bioRxiv2018

The landscape of intrinsic and evolved fluoroquinolone resistance in Acinetobacter baumannii includes suppression of drug-induced prophage replication

The emergence of fluoroquinolone resistance in nosocomial pathogens has restricted the clinical efficacy of this antibiotic class. In Acinetobacter baumannii, the majority of clinical isolates now show high-level resistance due to mutations in gyrA (DNA gyrase) and parC (Topo IV). To investigate the molecular basis for fluoroquinolone resistance, an exhaustive mutation analysis was performed in both drug sensitive and resistant strains to identify loci that alter the sensitivity of the organism to ciprofloxacin. To this end, parallel fitness tests of over 60,000 unique insertion mutations were performed in strains with various alleles in genes encoding the drug targets. The spectrum of mutations that altered drug sensitivity was found to be similar in the drug sensitive and double mutant gyrAparC background having resistance alleles in both genes. In contrast, introduction of a single gyrA resistance allele, resulting in preferential poisoning of Topo IV by ciprofloxacin, led to extreme alterations in the insertion mutation fitness landscape. The distinguishing feature of preferential Topo IV poisoning was induction of DNA synthesis in the region of two endogenous prophages, which appeared to occur in situ. Induction of the selective DNA synthesis in the gyrA background was also linked to enhanced activation of SOS response and heightened transcription of prophage genes relative to that observed in either the WT or gyrAparC double mutants. Therefore, the accumulation of mutations that result in the stepwise evolution of high ciprofloxacin resistance is tightly connected to suppression of hyperactivation of the SOS response and endogenous prophage DNA synthesis.\n\nImportanceFluoroquinolones have been extremely successful antibiotics. Their clinical efficacy derives from the ability to target multiple bacterial enzymes critical to DNA replication, the topoisomerases DNA gyrase and Topo IV. Unfortunately, mutations lowering drug affinity for both enzymes are now widespread, rendering these drugs ineffective for many pathogens. To undermine this form of resistance, we sought to understand how bacteria with target alterations differentially cope with fluoroquinolone exposures. We studied this problem in the nosocomial pathogen A. baumannii, which causes resistant, life-threating infections. Employing genome-wide approaches, we uncovered numerous pathways that could be exploited to lower fluoroquinolone resistance independently of target alteration. Remarkably, fluoroquinolone targeting of Topo IV in specific mutants caused dramatic prophage hyperinduction, a response that was muted in strains with DNA gyrase as the primary target. This work demonstrates that resistance evolution via target modification can profoundly modulate the antibiotic stress response, revealing potential resistance-associated liabilities.

microbiology↗