SarS and Rot are necessary for the repression of lukED and lukSF-PV in Staphylococcus aureus.

IMPORTANCE: The leukocidins play an important role in disarming the host immune system and promoting infection. While both SarS and Rot have been established as repressors of leukocidins, the importance of each repressor in infection is unclear. Here, we demonstrate that repression by SarS and Rot is not additive and show that in addition to upregulating expression of each other, they are also able to bind concurrently to the leukocidin promoters. These findings suggest that both repressors are necessary for maximal repression of lukED and lukSF-PV and illuminate another complex relationship among Staphylococcus aureus virulence regulators.

Unlatching of the stem domains in the Staphylococcus aureus pore-forming leukocidin LukAB influences toxin oligomerization.

Staphylococcus aureus (S. aureus) is a serious global pathogen that causes a diverse range of invasive diseases. S. aureus utilizes a family of pore-forming toxins, known as bi-component leukocidins, to evade the host immune response and promote infection. Among these is LukAB (leukocidin A/leukocidin B), a toxin that assembles into an octameric ß-barrel pore in the target cell membrane, resulting in host cell death. The established cellular receptor for LukAB is CD11b of the Mac-1 complex. Here, we show that hydrogen voltage-gated channel 1 is also required for the cytotoxicity of all major LukAB variants. We demonstrate that while each receptor is sufficient to recruit LukAB to the plasma membrane, both receptors are required for maximal lytic activity. Why LukAB requires two receptors, and how each of these receptors contributes to pore-formation remains unknown. To begin to resolve this, we performed an alanine scanning mutagenesis screen to identify mutations that allow LukAB to maintain cytotoxicity without CD11b. We discovered 30 mutations primarily localized in the stem domains of LukA and LukB that enable LukAB to exhibit full cytotoxicity in the absence of CD11b. Using crosslinking, electron microscopy, and hydroxyl radical protein footprinting, we show these mutations increase the solvent accessibility of the stem domain, priming LukAB for oligomerization. Together, our data support a model in which CD11b binding unlatches the membrane penetrating stem domains of LukAB, and this change in flexibility promotes toxin oligomerization.

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.

SarS Is a Repressor of Staphylococcus aureus Bicomponent Pore-Forming Leukocidins.

Staphylococcus aureus is a successful pathogen that produces a wide range of virulence factors that it uses to subvert and suppress the immune system. These include the bicomponent pore-forming leukocidins. How the expression of these toxins is regulated is not completely understood. Here, we describe a screen to identify transcription factors involved in the regulation of leukocidins. The most prominent discovery from this screen is that SarS, a known transcription factor which had previously been described as a repressor of alpha-toxin expression, was found to be a potent repressor of leukocidins LukED and LukSF-PV. We found that inactivating sarS resulted in increased virulence both in an ex vivo model using primary human neutrophils and in an in vivo infection model in mice. Further experimentation revealed that SarS represses leukocidins by serving as an activator of Rot, a critical repressor of toxins, as well as by directly binding and repressing the leukocidin promoters. By studying contemporary clinical isolates, we identified naturally occurring mutations in the sarS promoter that resulted in overexpression of sarS and increased repression of leukocidins in USA300 bloodstream clinical isolates. Overall, these data establish SarS as an important repressor of leukocidins and expand our understanding of how these virulence factors are being regulated in vitro and in vivo by S. aureus.

MRSA lineage USA300 isolated from bloodstream infections exhibit altered virulence regulation.

The epidemic community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 lineage has recently become a leading cause of hospital-associated bloodstream infections (BSIs). Here, we leveraged this recent introduction into hospitals and the limited genetic variation across USA300 isolates to identify mutations that contribute to its success in a new environment. We found that USA300 BSI isolates exhibit altered virulence regulation. Using comparative genomics to delineate the genes involved in this phenotype, we discovered repeated and independent mutations in the transcriptional regulator sarZ. Mutations in sarZ resulted in increased virulence of USA300 BSI isolates in a murine model of BSI. The sarZ mutations derepressed the expression and production of the surface protein ClfB, which was critical for the pathogenesis of USA300 BSI isolates. Altogether, these findings highlight ongoing evolution of a major MRSA lineage and suggest USA300 strains can optimize their fitness through altered regulation of virulence.

Genetic variation of staphylococcal LukAB toxin determines receptor tropism.

Staphylococcus aureus has evolved into diverse lineages, known as clonal complexes (CCs), which exhibit differences in the coding sequences of core virulence factors. Whether these alterations affect functionality is poorly understood. Here, we studied the highly polymorphic pore-forming toxin LukAB. We discovered that the LukAB toxin variants produced by S. aureus CC30 and CC45 kill human phagocytes regardless of whether CD11b, the previously established LukAB receptor, is present, and instead target the human hydrogen voltage-gated channel 1 (HVCN1). Biochemical studies identified the domain within human HVCN1 that drives LukAB species specificity, enabling the generation of humanized HVCN1 mice with enhanced susceptibility to CC30 LukAB and to bloodstream infection caused by CC30 S. aureus strains. Together, this work advances our understanding of an important S. aureus toxin and underscores the importance of considering genetic variation in characterizing virulence factors and understanding the tug of war between pathogens and the host.

The Spike D614G mutation increases SARS-CoV-2 infection of multiple human cell types.

A novel variant of the SARS-CoV-2 virus carrying a point mutation in the Spike protein (D614G) has recently emerged and rapidly surpassed others in prevalence. This mutation is in linkage disequilibrium with an ORF1b protein variant (P314L), making it difficult to discern the functional significance of the Spike D614G mutation from population genetics alone. Here, we perform site-directed mutagenesis on wild-type human-codon-optimized Spike to introduce the D614G variant. Using multiple human cell lines, including human lung epithelial cells, we found that the lentiviral particles pseudotyped with Spike D614G are more effective at transducing cells than ones pseudotyped with wild-type Spike. The increased transduction with Spike D614G ranged from 1.3- to 2.4-fold in Caco-2 and Calu-3 cells expressing endogenous ACE2 and from 1.5- to 7.7-fold in A549ACE2 and Huh7.5ACE2 overexpressing ACE2. Furthermore, trans-complementation of SARS-CoV-2 virus with Spike D614G showed an increased infectivity in human cells. Although there is minimal difference in ACE2 receptor binding between the D614 and G614 Spike variants, the G614 variant is more resistant to proteolytic cleavage, suggesting a possible mechanism for the increased transduction.

The Spike D614G mutation increases SARS-CoV-2 infection of multiple human cell types.

A novel isolate of the SARS-CoV-2 virus carrying a point mutation in the Spike protein (D614G) has recently emerged and rapidly surpassed others in prevalence. This mutation is in linkage disequilibrium with an ORF1b protein variant (P314L), making it difficult to discern the functional significance of the Spike D614G mutation from population genetics alone. Here, we perform site-directed mutagenesis to introduce the D614G variant and show that in multiple cell lines, including human lung epithelial cells, that the D614G mutation is up to 8-fold more effective at transducing cells than wild-type. We demonstrate increased infection using both Spike-pseudotyped lentivirus and intact SARS-CoV-2 virus. Although there is minimal difference in ACE2 receptor binding between the Spike variants, we show that the G614 variant is more resistant to proteolytic cleavage in vitro and in human cells, suggesting a possible mechanism for the increased transduction. This result has important implications for the efficacy of Spike-based vaccines currently under development in protecting against this recent and highly-prevalent SARS-CoV-2 isolate.

High resources and infectious disease facilitate invasion by a freshwater crustacean.

It is well-established that both resources and infectious disease can influence species invasions, but little is known regarding interactive effects of these two factors. We performed a series of experiments to understand how resources and parasites can jointly affect the ability of a freshwater invasive zooplankton to establish in a population of a native zooplankton. In a life history trial, we found that both species increased offspring production to the same degree as algal resources increased, suggesting that changes in resources would have similar effects on both species. In a microcosm experiment simulating an invasion, we found that the invasive species reached its highest densities when there was a combination of both high resources and the presence of a shared parasite, but not for each of these conditions alone (i.e., a significant resource x parasite interaction). This result can be explained by changes in native host population density; high resource levels initially led to an increase in the density of the native host, which caused larger epidemics when the parasite was present. This high infection prevalence caused a subsequent reduction in native host density, increasing available resources and allowing the invasive species to establish relatively dense populations. Thus, in this system, native communities with a combination of high resource levels and parasitism may be the most vulnerable to invasions. More generally, our results suggest that parasitism and resource availability can have interactive, non-additive effects on the outcome of invasions.