Loessner, M. J. | bioRxiv Science

bioRxiv2026

A teichoic acid-like wall modification associated with immune suppression is socially regulated in Streptococcus pyogenes

Streptococcus pyogenes (Group A Streptococcus, GAS) is a human-restricted pathogen with a range of clinical manifestations and worldwide prevalence. The GAS Rgg2/Rgg3 quorum sensing (QS) system, a cell-to-cell communication network, modifies the cell surface resulting in increased lysozyme resistance, biofilm formation, and expression of the qim operon that is responsible for modulation of innate immune responses in macrophages. The operon encodes 10 genes with predicted homology to enzymes involved in bacterial cell surface-associated carbohydrate and teichoic acid biosynthesis pathways. Comparing extracts of GAS cell wall polysaccharides between wildtype and operon mutants determined that the QS-induced genes modify the S. pyogenes cell surface by adding a wall teichoic acid-like moiety of N-acetylglucosamine-linked ribitol (GlcNAc-Rbo). A fluorescently labeled phage receptor-binding protein, RBP-13-GFP, that recognizes GlcNAc-decorated ribitol phosphate repeats, bound to the GAS surface only when qim expression was induced. Deletion of the qim operon eliminated RBP-13-GFP binding, diminished bacterial colonization, and significantly attenuated GAS pathogenesis in a murine skin infection model. These findings indicate that GAS has evolved a strategy to evade innate immune response by presenting a previously unknown carbohydrate moiety upon quorum sensing. IMPORTANCEStreptococcus pyogenes is a major human pathogen, responsible for diverse clinical manifestations of both superficial and invasive infections and can lead to post-infection sequelae like rheumatic heart disease whose prevalence on a global scale rivals the most serious pathogens. Invasive S. pyogenes infections are currently on the rise worldwide, notably correlating with increasing pediatric cases of scarlet fever and enhancing the concern for long term complications. There is much that remains unknown about S. pyogenes virulence and pathogenicity, and studies focused on understanding basic systems regulating virulence factors could lead to better therapeutics and translational research. We show here one such example, where a bacterial communication system regulating a virulence mechanism relevant to in vivo infection confers the ability to alter the host innate immune response. We find that modifications to the cell wall arise when this virulence system is activated that has a direct role in host-pathogen interactions. Further research into this system could provide a mechanism for disruption and serve to treat S. pyogenes infection.

microbiology↗

bioRxiv2024

Computational pipeline for targeted integration and variable payload expression for bacteriophage engineering.

Bacteriophages offer a promising alternative to conventional antimicrobial treatments, particularly in cases where such treatments have proven ineffective. While naturally occurring phages serve as viable options for phage therapy, advances in synthetic biology and genome engineering enable precise modifications to phages to enhance their therapeutic potential. One such approach is the introduction of antimicrobial genetic payloads into the phage genome. Conventional practice is to integrate such payloads behind genes expressed at very high levels late in the infection cycle, such as the major capsid gene (cps). Nevertheless, phages engineered to contain antimicrobial payloads are often difficult to obtain. For instance, the high expression of toxic payloads can prematurely halt host metabolism, leading to the failure in assembling viable phage progeny. To potentially expand the range of genes viable as genetic payloads, we developed a method to identify intergenic loci with favorable expression levels. We used the machine learning (ML)-based promoter prediction algorithm PhagePromoter to identify these loci. We then used this information to design a computationally-assisted engineering pipeline for the integration of genomic payloads at these locations. We validated our approach experimentally, engineering phages with bioluminescent reporter payloads at various predicted loci. We used the well characterized, strictly lytic, Staphylococcus aureus -infecting bacteriophage, K, as an engineering scaffold and employed homologous recombination to engineer three recombinant phages containing the reporter payload at different predicted loci throughout the genome. The recombinant phages exhibited expression levels consistent with our computational predictions and showed temporal expression patterns corresponding to their genomic locations in early, middle, or late gene clusters. Our study underscores the potential of integrating computational tools with classical sequence analysis to streamline the phage engineering process. This approach not only facilitates the rational design of phages with targeted payload insertions but also paves the way for high-throughput, automated phage engineering, fostering a new era of personalized phage therapy.

synthetic biology↗

bioRxiv2024

CRISPR-Cas9 enables efficient genome engineering of the strictly lytic, broad host-range staphylococcal bacteriophage K

Staphylococcus aureus is an important opportunistic pathogen, responsible for a range of diseases that often prove challenging to treat due to resistance to methicillin, vancomycin, and other antimicrobials. Bacteriophages present a promising alternative to target such pathogens, particularly when conventional drugs are ineffective. The antimicrobial efficacy of phage therapeutics can be further improved through genetic engineering. Among S. aureus phages, members of the Twortvirinae subfamily, characterized by their strictly lytic nature and broad host range, are considered the most promising therapeutic candidates. However, their large genome sizes make them notoriously difficult to engineer. In this study, we utilized Twortvirus K as a model to develop an efficient phage engineering platform, leveraging homologous recombination and CRISPR-Cas9-assisted counterselection. As proof of principle, this platform was utilized to construct a nanoluciferase (nluc)-encoding reporter phage (K::nluc) and tested as a preliminary, bioluminescence-based approach for identifying viable Staphylococcus cells. Independent of their phage-resistance profile, 100% of tested clinical S. aureus isolates emitted bioluminescence upon K::nluc challenge. This diagnostic assay was further adapted to complex matrices such as human whole blood and bovine raw milk, simulating S. aureus detection scenarios in bacteremia and bovine mastitis. Beyond reporter phage-based diagnostics, our engineering technology opens avenues for the design and engineering of therapeutic Twortvirinae phages to combat drug-resistant S. aureus strains.

bioengineering↗

bioRxiv2024

Heterodimerization of Endolysin Isoforms During Bacterial Infection by Staphylococcal Phage {varphi}2638A

AO_SCPLOWBSTRACTC_SCPLOWBacteriophage endolysins targeting Gram-positive bacteria typically feature a modular architecture of one or more enzymatically active domains (EADs) and cell wall binding domains (CBDs). Several endolysins also feature internal translational start sites (iTSSs) that produce short variant (SV) isoforms alongside the full-length (FL) endolysin. While the lytic activity of endolysins and their isoforms has been extensively studied as exogenous agents, the purpose behind producing the SV isoform during the phage infection cycle remains to be explored. In this study, we used staphylococcal phage {varphi}2638A as a model to determine the interplay between its full-length endolysin, Ply2638A, and its SV isoform during phage infection. X-ray crystallography structures and AlphaFold-generated models enabled elucidation of individual functions of the M23 endopeptidase, central amidase, and SH3b domains of Ply2638A. Production of the SV isoform (amidase and SH3b) was confirmed during phage infection and shown to form a heterodimer complex with Ply2638A via inter-amidase domain interactions. Using genetically engineered phage variants, we show that production of both isoforms provides an advantage during phage infection as phages producing only one isoform presented impaired lytic activity, which could be partly restored through recombinant protein complementation of the missing isoform. Importantly, when applied as an antimicrobial protein against Staphylococcus aureus in culture, the activity of Ply2638A remained constant regardless of SV isoform complementation. Drawing from our findings, we propose that SV isoform production provides its biological advantage upon endolysin entry to the periplasmic space to ensure optimal peptidoglycan degradation prior to cell wall lysis and progeny phage release.

molecular biology↗

bioRxiv2022

Engineered reporter phages for rapid detection of Escherichia coli, Klebsiella spp., and Enterococcus spp. in urine

The rapid detection and species-level differentiation of bacterial pathogens facilitates antibiotic stewardship and improves disease management. Here, we develop a rapid bacteriophage-based diagnostic assay to detect the most prevalent pathogens causing urinary tract infections: Escherichia coli, Klebsiella spp., and Enterococcus spp. For each uropathogen, two virulent phages were genetically engineered to express a nanoluciferase reporter gene upon host infection. Using 206 patient urine samples, reporter phage-induced bioluminescence was quantified to identify bacteriuria and the assay was benchmarked against conventional urinalysis. Overall, E. coli, Klebsiella spp., and Enterococcus spp. were each detected with high sensitivity (68%, 78%, 85%), specificity (99%, 99%, 99%), and accuracy (90%, 94%, 96%) at a resolution of [>=]103 CFU/ml within 5 h. We further demonstrate how bioluminescence in urine can be used to predict phage antibacterial activity, demonstrating the future potential of reporter phages as companion diagnostics that guide patient-phage matching prior to therapeutic phage application.

microbiology↗

bioRxiv2022

Gram-positive bacteria evade phage predation through endolysin-mediated L-form conversion

Bacteriophages kill bacteria by osmotic lysis towards the end of the lytic cycle. In the case of Gram-positive bacteria, peptidoglycan-degrading endolysins released at the end of infection cycle cause explosive cell lysis not only of the infected host, but can also attack non-infected bystander cells. Here, we show that in osmotically stabilized environments, Listeria monocytogenes can evade phage predation by transient conversion to a cell wall-deficient L-form state. This L-form escape is triggered by endolysins disintegrating the cell wall from without, leading to turgor-driven extrusion of wall-deficient, yet viable L-form cells. Remarkably, in absence of phage predation, we show that L-forms can quickly revert to the walled state. These findings suggest that L-form conversion represents a population-level persistence mechanism to evade complete eradication by phage attack. Importantly, we also demonstrate phage-mediated L-form switching of the urinary tract pathogen Enterococcus faecalis in human urine, which underscores that this escape route may be widespread and has important implications for phage- and endolysin-based therapeutic interventions.

microbiology↗

bioRxiv2022

Enhancing bacteriophage therapeutics through in situ production and release of heterologous antimicrobial effectors

Bacteriophages operate via pathogen-specific mechanisms of action distinct from conventional, broad-spectrum antibiotics and are emerging as promising alternatives. However, phage-mediated killing is often limited by bacterial resistance development (1,2). Here, we engineer phages for target-specific effector gene delivery and host-dependent production of colicin-like bacteriocins and cell wall hydrolases. Using urinary tract infection (UTI) as a model, we show how heterologous effector phage therapeutics (HEPTs) suppress resistance and improve uropathogen killing by dual phage- and effector-mediated targeting. Moreover, we designed HEPTs to control polymicrobial uropathogen communities through production of effectors with cross-genus activity. Using a phage-based companion diagnostic (3), we identified potential HEPT responder patients and treated their urine ex vivo. Compared to wildtype phage, a colicin E7-producing HEPT demonstrated superior control of patient E. coli bacteriuria. Arming phages with heterologous effectors paves the way for successful UTI treatment and represents a versatile tool to enhance and adapt phage-based precision antimicrobials.

bioengineering↗

bioRxiv2021

Glucose decoration on wall-teichoic acid is required for phage adsorption and InlB-mediated virulence in Listeria ivanovii

Listeria ivanovii (Liv) is an intracellular Gram-positive pathogen that primarily infects ruminants, but also occasionally causes enteric infections in humans. Albeit rare, this bacterium possesses the capacity to cross the intestinal epithelium of humans, similar to its more frequently pathogenic cousin, Listeria monocytogenes (Lmo). Recent studies in Lmo have shown that specific glycosyl modifications on the cell wall-associated glycopolymers (termed wall-teichoic acid, or WTA) of Lmo are responsible for bacteriophage adsorption and retention of the major virulence factor, Internalin B (InlB). However, the relationship between InlB and WTA in Liv remains unclear. Here, we report the identification of the unique gene, liv1070 that encodes a putative glucosyltransferase in the polycistronic WTA gene cluster of the Liv WSLC 3009 genome. We found that in-frame deletion of liv1070 led to loss of the glucose substitution on WTA, as revealed by UPLC-MS analysis. Interestingly, the glucose-deficient mutant became resistant to phage B025 infection due to an inability of the phage to adsorb to the bacterial surface, a binding process mediated by the receptor-binding protein B025_Gp17. As expected, deletion of liv1070 led to loss of InlB retention to the bacterial cell wall, which corresponded to a drastic decrease in cellular invasion. Genetic complementation of liv1070 restored the characteristic phenotypes, including glucose decoration, phage adsorption, and cellular invasion. Taken together, our data demonstrate that an interplay between phage, bacteria, and host cells also exists in Listeria ivanovii, suggesting the trade-off between phage resistance and virulence attenuation may be a general feature in the Listeria genus. ImportanceListeria ivanovii is a Gram-positive bacterial pathogen known to cause enteric infection in rodents and ruminants, and occasionally in immunocompromised humans. Recent investigations revealed that, in its better-known cousin Listeria monocytogenes, strains develop resistance to bacteriophage attack due to loss of glycosylated surface receptors, which subsequently resulting in disconnection of one of the bacteriums major virulence factors, InlB. However, the situation in L. ivanovii remains unclear. Here, we show that L. ivanovii acquires phage resistance following deletion of a unique glycosyltransferase. This deletion also leads to dysfunction of InlB, making the resulting strain unable to invade host cells. Overall, this study suggests that the interplay between phage, bacteria and the host may be a feature common to the Listeria genus.

microbiology↗

bioRxiv2021

Glucose decoration on wall-teichoic acid is required for phage adsorption and InlB-mediated virulence in Listeria ivanovii

Listeria ivanovii (Liv) is an intracellular Gram-positive pathogen that primarily infects ruminants, but also occasionally causes enteric infections in humans. Albeit rare, this bacterium possesses the capacity to cross the intestinal epithelium of humans, similar to its more frequently pathogenic cousin, Listeria monocytogenes (Lmo). Recent studies in Lmo have shown that specific glycosyl modifications on the cell wall-associated glycopolymers (termed wall-teichoic acid, or WTA) of Lmo are responsible for bacteriophage adsorption and retention of the major virulence factor, Internalin B (InlB). However, the relationship between InlB and WTA in Liv remains unclear. Here, we report the identification of the unique gene, liv1070 that encodes a putative glucosyltransferase in the polycistronic WTA gene cluster of the Liv WSLC 3009 genome. We found that in-frame deletion of liv1070 led to loss of the glucose substitution on WTA, as revealed by UPLC-MS analysis. Interestingly, the glucose-deficient mutant became resistant to phage B025 infection due to an inability of the phage to adsorb to the bacterial surface, a binding process mediated by the receptor-binding protein B025_Gp17. As expected, deletion of liv1070 led to loss of InlB retention to the bacterial cell wall, which corresponded to a drastic decrease in cellular invasion. Genetic complementation of liv1070 restored the characteristic phenotypes, including glucose decoration, phage adsorption, and cellular invasion. Taken together, our data demonstrate that an interplay between phage, bacteria, and host cells also exists in Listeria ivanovii, suggesting the trade-off between phage resistance and virulence attenuation may be a general feature in the Listeria genus. ImportanceListeria ivanovii is a Gram-positive bacterial pathogen known to cause enteric infection in rodents and ruminants, and occasionally in immunocompromised humans. Recent investigations revealed that, in its better-known cousin Listeria monocytogenes, strains develop resistance to bacteriophage attack due to loss of glycosylated surface receptors, which subsequently resulting in disconnection of one of the bacteriums major virulence factors, InlB. However, the situation in L. ivanovii remains unclear. Here, we show that L. ivanovii acquires phage resistance following deletion of a unique glycosyltransferase. This deletion also leads to dysfunction of InlB, making the resulting strain unable to invade host cells. Overall, this study suggests that the interplay between phage, bacteria and the host may be a feature common to the Listeria genus.

microbiology↗

bioRxiv2020

Bacillus subtilis YngB is a UTP-glucose-1-phosphate uridylyltransferase contributing to wall teichoic acid modification and glycolipid formation during anaerobic growth

UTP-glucose-1-phosphate uridylyltransferases (UGPases) are enzymes that produce UDP-glucose from UTP and glucose-1-phosphate. In Bacillus subtilis 168, UDP-glucose is required for the decoration of wall teichoic acid (WTA) with glucose residues and the formation of glucolipids. The B. subtilis UGPase GtaB is essential for UDP-glucose production under standard aerobic growth conditions, and gtaB mutants display severe growth and morphological defects. However, bioinformatics predictions indicate that two other UGPases, are present in B. subtilis. Here, we investigated the function of one of them named YngB. The crystal structure of YngB revealed that the protein has the typical fold and all necessary active site features of a functional UGPase. Furthermore, UGPase activity could be demonstrated in vitro using UTP and glucose-1-phosphate as substrates. Expression of YngB from a synthetic promoter in a B. subtilis gtaB mutant resulted in the reintroduction of glucose residues on WTA and production of glycolipids, demonstrating that the enzyme can function as UGPase in vivo. When wild-type and mutant B. subtilis strains were grown under anaerobic conditions, YngB-dependent glycolipid production and glucose decorations on WTA could be detected, revealing that YngB is expressed from its native promoter under anaerobic condition. Based on these findings, along with the structure of the operon containing yngB and the transcription factor thought to be required for its expression, we propose that besides WTA, potentially other cell wall components might be decorated with glucose residues during oxygen limited growth condition.

microbiology↗

bioRxiv2020

Light-mediated discovery of surfaceome nanoscale organization and intercellular receptor interaction networks

Delineating the molecular nanoscale organization of the surfaceome is pre-requisite for understanding cellular signaling. Technologies for mapping the spatial relationships of cell surface receptors and their extracellular signaling synapses would open up theranostic opportunities and the possibility to engineer extracellular signaling. Here, we developed an optoproteomic technology termed LUX-MS that exploits singlet oxygen generators (SOG) for the light-triggered identification of acute protein interactions on living cells. Using SOG-coupled antibodies, small molecule-drugs, biologics and intact viral particles, we show that not only ligand-receptor interactions can be decoded across organisms, but also the surfaceome receptor nanoscale organization ligands engage in with direct implications for drug action. Furthermore, investigation of functional immunosynapses revealed that intercellular signaling inbetween APCs and CD8+ T cells can be mapped now providing insights into T cell activation with spatiotemporal resolution. LUX-MS based decoding of surfaceome signaling architectures provides unprecedented molecular insights for the rational development of theranostic strategies.

systems biology↗

bioRxiv2019

Uncovering a hidden diversity: optimized protocols for the extraction of bacteriophages from soil

BackgroundBacteriophages are the most numerous biological entities on earth and play a crucial role in shaping microbial communities. Investigating the bacteriophage community from soil samples will shed light not only on the yet largely unknown phage diversity, but also may result in novel insights into phage biology and functioning. Unfortunately, the study of soil viromes lags far behind any other ecological model system, due to the heterogeneous soil matrix that rises major technical difficulties in the extraction process. Resolving these technical challenges and establishing a standardized extraction protocol is therefore a fundamental prerequisite for replicable results and comparative virome studies.\n\nResultsWe here report the optimization of protocols for extraction of bacteriophage DNA from soil preceding metagenomic analysis such that the protocol can equally be harnessed for phage isolation. As an optimization strategy, soil samples were spiked with a viral community consisting of phages from different families (106 PFU/g soil): Listeria phage {Phi}A511 (Myovirus), Staphylococcus phage {Phi}2638A{Delta}LCR (Siphovirus), and Escherichia phage {Phi}T7 (Podovirus). The efficacy of bacteriophage (i) elution, (ii) filtration, (iii) concentration, and (iv) DNA extraction methods was tested. Successful extraction routes were selected based on spiked phage recovery and low bacterial 16S rRNA gene contaminants. Natural agricultural soil viromes were then extracted with the optimized methods and shotgun sequenced. Our approach yielded sufficient amounts of inhibitor-free viral DNA for non-amplification dependent sequencing and low 16S rRNA gene contamination levels ([≤] 0.2 {per thousand}). Compared to previously published protocols, the number of bacterial read contamination was decreased by 65 %. In addition, 468 novel circularized soil phage genomes in size up to 235 kb were obtained from over 29,000 manually identified viral contigs, promising the discovery of a large, previously inaccessible viral diversity.\n\nConclusionWe have shown a dramatically enhanced extraction of the soil phage community by protocol optimization that has proven robustness in both a culture-depended as well as through metaviromic analysis. Our huge data set of manually curated soil viral contigs roughly doubles the amount of currently available soil virome data, and provide insights into the yet largely undescribed soil viral sequence space.

microbiology↗

bioRxiv2019

A proteogenomic resource enabling integrated analysis of Listeria genotype-proteotype-phenotype relationships

Listeria monocytogenes is an opportunistic foodborne pathogen responsible for listeriosis, a potentially fatal foodborne disease. Many different Listeria strains and serotypes exist, but a proteogenomic resource that bridges the gap in our molecular understanding of the relationships between the Listeria genotypes and phenotypes via proteotypes is still missing. Here we devised a next-generation proteogenomics strategy that enables the community to rapidly proteotype Listeria strains and relate this information back to the genotype. Based on sequencing and de novo assembly of the two most commonly used Listeria model strains, EGD-e and ScottA, we established two comprehensive Listeria proteogenomic databases. A genome comparison established core- and strain-specific genes potentially responsible for virulence differences. Next, we established a DIA/SWATH-based proteotyping strategy, including a new and robust sample preparation workflow, that enables the reproducible, sensitive, and relative quantitative measurement of Listeria proteotypes. This reusable and publically available DIA/SWATH library covers 70% of open reading frames of Listeria and represents the most extensive spectral library for Listeria proteotype analysis to date. We used these two new resources to investigate the Listeria proteotype in states mimicking the upper gastrointestinal passage. Exposure of Listeria to bile salts at 37 {degrees}C, which simulates conditions encountered in the duodenum, showed significant proteotype perturbations including an increase of FlaA, the structural protein of flagella. Given that Listeria is known to lose its flagella above 30 {degrees}C, this was an unexpected finding. The formation of flagella, which might have implications on infectivity, was validated by parallel reaction monitoring and light and scanning electron microscopy. flaA transcript levels were not significantly different with and without exposure to bile salts at 37 {degrees}C, suggesting regulation at the post-transcriptional level. Together, these analyses provide a comprehensive proteogenomic resource and toolbox for the Listeria community enabling the analysis of Listeria genotype-proteotype-phenotype relationships.

systems biology↗