Characterization of tigurilysin, a novel human CD59-specific cholesterol-dependent cytolysin, reveals a role for host specificity in augmenting toxin activity.

Cholesterol-dependent cytolysins (CDCs) are a large family of pore-forming toxins, produced by numerous Gram-positive pathogens. CDCs depend on host membrane cholesterol for pore formation; some CDCs also require surface-associated human CD59 (hCD59) for binding, conferring specificity for human cells. We purified a recombinant version of a putative CDC encoded in the genome of Streptococcus oralis subsp. tigurinus, tigurilysin (TGY), and used CRISPR/Cas9 to construct hCD59 knockout (KO) HeLa and JEG-3 cell lines. Cell viability assays with TGY on wild-type and hCD59 KO cells showed that TGY is a hCD59-dependent CDC. Two variants of TGY exist among S. oralis subsp. tigurinus genomes, only one of which is functional. We discovered that a single amino acid change between these two TGY variants determines its activity. Flow cytometry and oligomerization Western blots revealed that the single amino acid difference between the two TGY isoforms disrupts host cell binding and oligomerization. Furthermore, experiments with hCD59 KO cells and cholesterol-depleted cells demonstrated that TGY is fully dependent on both hCD59 and cholesterol for activity, unlike other known hCD59-dependent CDCs. Using full-length CDCs and toxin constructs differing only in the binding domain, we determined that having hCD59 dependence leads to increased lysis efficiency, conferring a potential advantage to organisms producing hCD59-dependent CDCs.

Characterization of Tigurilysin, a Novel Human CD59-Specific Cholesterol-Dependent Cytolysin, Reveals a Role for Host Specificity in Augmenting Toxin Activity.

Cholesterol dependent cytolysins (CDCs) are a large family of pore forming toxins, produced by numerous gram-positive pathogens. CDCs depend on host membrane cholesterol for pore formation; some CDCs also require surface associated human CD59 (hCD59) for binding, conferring specificity for human cells. We purified a recombinant version of a putative CDC encoded in the genome of Streptococcus oralis subsp. tigurinus , tigurilysin (TGY), and used CRISPR/Cas9 to construct hCD59 knockout (KO) HeLa and JEG-3 cell lines. Cell viability assays with TGY on WT and hCD59 KO cells showed that TGY is a hCD59-dependent CDC. Two variants of TGY exist among S. oralis subsp. tigurinus genomes, only one of which is functional. We discovered that a single amino acid change between these two TGY variants determines its activity. Flow cytometry and oligomerization western blots revealed that the single amino acid difference between the two TGY isoforms disrupts host cell binding and oligomerization. Furthermore, experiments with hCD59 KO cells and cholesterol depleted cells demonstrated that TGY is fully dependent on both hCD59 and cholesterol for activity, unlike other known hCD59-dependent CDCs. Using full-length CDCs and toxin constructs differing only in the binding domain, we determined that having hCD59-dependence leads to increased lysis efficiency, conferring a potential advantage to organisms producing hCD59-dependent CDCs. IMPORTANCE: Cholesterol dependent cytolysins (CDCs) are produced by a variety of disease-causing bacteria, and may play a significant role in pathogenesis. Understanding CDC mechanisms of action provides useful information for developing anti-virulence strategies against bacteria that utilize CDCs and other pore-forming toxins in pathogenesis. This study describes for the first time a novel human-specific CDC with an atypical pore forming mechanism compared to known CDCs. In addition, this study demonstrates that human-specificity potentially confers increased lytic efficiency to CDCs. These data provide a possible explanation for the selective advantage of developing hCD59-dependency in CDCs and the consequent host restriction.

Genome-Wide CRISPR-Cas9 Screen Does Not Identify Host Factors Modulating Streptococcus agalactiae ß-Hemolysin/Cytolysin-Induced Cell Death.

Pore-forming toxins (PFTs) are commonly produced by pathogenic bacteria, and understanding them is key to the development of virulence-targeted therapies. Streptococcus agalactiae, or group B Streptococcus (GBS), produces several factors that enhance its pathogenicity, including the PFT ß-hemolysin/cytolysin (ßhc). Little is understood about the cellular factors involved in ßhc pore formation. We conducted a whole-genome CRISPR-Cas9 forward genetic screen to identify host genes that might contribute to ßhc pore formation and cell death. While the screen identified the established receptor, CD59, in control experiments using the toxin intermedilysin (ILY), no clear candidate genes were identified that were required for ßhc-mediated lethality. Of the top targets from the screen, two genes involved in membrane remodeling and repair represented candidates that might modulate the kinetics of ßhc-induced cell death. Upon attempted validation of the results using monoclonal cell lines with targeted disruption of these genes, no effect on ßhc-mediated cell lysis was observed. The CRISPR-Cas9 screen results are consistent with the hypothesis that ßhc does not require a single nonessential host factor to mediate target cell death. IMPORTANCE CRISPR-Cas9 forward genetic screens have been used to identify host cell targets required by bacterial toxins. They have been used successfully to both verify known targets and elucidate novel host factors required by toxins. Here, we show that this approach fails to identify host factors required for cell death due to ßhc, a toxin required for GBS virulence. These data suggest that ßhc may not require a host cell receptor for toxin function or may require a host receptor that is an essential gene and would not be identified using this screening strategy.

Group B Streptococcus Capsular Serotype Alters Vaginal Colonization Fitness.

BACKGROUND: Group B Streptococcus (GBS) remains a leading cause of infant morbidity and mortality. A candidate vaccine targets 6 GBS serotypes, offering a potential alternative to intrapartum antibiotic prophylaxis to reduce disease burden. However, our understanding of the contributions of specific capsule types to GBS colonization and disease remains limited. METHODS: Using allelic exchange, we generated isogenic GBS strains differing only in the serotype-determining region in 2 genetic backgrounds, including the hypervirulent clonal complex (CC) 17. Using a murine model of vaginal cocolonization, we evaluated the roles of the presence of capsule and of expression of specific capsular types in GBS vaginal colonization fitness independent of other genetic factors. RESULTS: Encapsulated wild-type strains COH1 (CC17, serotype III) and A909 (non-CC17, serotype Ia) outcompeted isogenic acapsular mutants in murine vaginal cocolonization. COH1 wild type outcompeted A909. Notably, expression of type Ia capsule conferred an advantage over type III capsule in both genetic backgrounds. CONCLUSIONS: Specific capsule types may provide an advantage in GBS vaginal colonization in vivo. However, success of certain GBS lineages, including CC17, likely involves both capsule and noncapsule genetic elements. Capsule switching in GBS, a potential outcome of conjugate vaccine programs, may alter colonization fitness or pathogenesis.

Rewiring of Glutamine Metabolism Is a Bioenergetic Adaptation of Human Cells with Mitochondrial DNA Mutations.

Using molecular, biochemical, and untargeted stable isotope tracing approaches, we identify a previously unappreciated glutamine-derived α-ketoglutarate (αKG) energy-generating anaplerotic flux to be critical in mitochondrial DNA (mtDNA) mutant cells that harbor human disease-associated oxidative phosphorylation defects. Stimulating this flux with αKG supplementation enables the survival of diverse mtDNA mutant cells under otherwise lethal obligatory oxidative conditions. Strikingly, we demonstrate that when residual mitochondrial respiration in mtDNA mutant cells exceeds 45% of control levels, αKG oxidative flux prevails over reductive carboxylation. Furthermore, in a mouse model of mitochondrial myopathy, we show that increased oxidative αKG flux in muscle arises from enhanced alanine synthesis and release into blood, concomitant with accelerated amino acid catabolism from protein breakdown. Importantly, in this mouse model of mitochondriopathy, muscle amino acid imbalance is normalized by αKG supplementation. Taken together, our findings provide a rationale for αKG supplementation as a therapeutic strategy for mitochondrial myopathies.