Virulence characteristics of Gram-positive bacteria isolated from diabetic foot ulcers.

Diabetic wound infections including diabetic foot ulcers (DFUs) are a major global health concern and a leading cause of non-traumatic amputations. Numerous bacterial species establish infection in DFUs, and treatment with antibiotics often fails due to widespread antibiotic resistance and biofilm formation. Determination of bacterial species that reside in DFU and their virulence potential is critical to inform treatment options. Here, we isolate bacteria from debridement tissues from patients with diabetes at the University of Colorado Anschutz Medical Center. The most frequent species were Gram-positive including Enterococcus faecalis, Staphylococcus aureus, and Streptococcus agalactiae, also known as Group B Streptococcus (GBS). Most tissues had more than one species isolated with E. faecalis and GBS frequently occurring in polymicrobial infection with S. aureus. S. aureus was the best biofilm producing species with E. faecalis and GBS isolates exhibiting little to no biofilm formation. Antibiotic susceptibility varied amongst strains with high levels of penicillin resistance amongst S. aureus, clindamycin resistance amongst GBS and intermediate vancomycin resistance amongst E. faecalis. Finally, we utilized a murine model of diabetic wound infection and found that the presence of S. aureus led to significantly higher recovery of GBS and E. faecalis compared to mice challenged in mono-infection.

Slower but more accurate mental rotation performance in aphantasia linked to differences in cognitive strategies.

Mental rotation tasks are frequently used as standard measures of mental imagery. However, aphantasia research has brought such use into question. Here, we assessed a large group of individuals who lack visual imagery (aphantasia) on two mental rotation tasks: a three-dimensional block-shape, and a human manikin rotation task. In both tasks, those with aphantasia had slower, but more accurate responses than controls. Both groups demonstrated classic linear increases in response time and error-rate as functions of angular disparity. In the three-dimensional block-shape rotation task, a within-group speed-accuracy trade-off was found in controls, whereas faster individuals in the aphantasia group were also more accurate. Control participants generally favoured using object-based mental rotation strategies, whereas those with aphantasia favoured analytic strategies. These results suggest that visual imagery is not crucial for successful performance in classical mental rotation tasks, as alternative strategies can be effectively utilised in the absence of holistic mental representations.

Revisiting the blind mind: Still no evidence for sensory visual imagery in individuals with aphantasia.

The inability to visualise was given the name aphantasia in 2015 by Zeman and colleagues. In 2018 we published research showing that fifteen individuals who self-identified as having aphantasia also demonstrated a lack of sensory visual imagery when undergoing the binocular rivalry imagery paradigm, suggesting more than just a metacognitive difference. Here we update these findings with over fifty participants with aphantasia and show that there is evidence for a lack of sensory imagery in aphantasia. How the binocular rivalry paradigm scores relate to the vividness of visual imagery questionnaire (VVIQ) and how aphantasia can be confirmed is discussed.

Androgens at the skin surface regulate S. aureus pathogenesis through the activation of agr quorum sensing.

Staphylococcus aureus, the most frequent cause of skin infections, is more common in men than women and selectively colonizes the skin during inflammation. Yet, the specific cues that drive infection in these settings remain unclear. Here we show that the host androgens testosterone and dihydrotestosterone promote S. aureus pathogenesis and skin infection. Without the secretion of these hormones, skin infection in vivo is limited. Testosterone activates S. aureus virulence in a concentration dependent manner through stimulation of the agr quorum sensing system, with the capacity to circumvent other inhibitory signals in the environment. Taken together, our work defines a previously uncharacterized inter-kingdom signal between the skin and the opportunistic pathogen S. aureus and identifies the mechanism of sex-dependent differences in S. aureus skin infection. One-Sentence Summary: Testosterone promotes S. aureus pathogenesis through activation of the agr quorum sensing system.

The Staphylococcus aureus CamS lipoprotein is a repressor of toxin production that shapes host-pathogen interaction.

Lipoproteins of the opportunistic pathogen Staphylococcus aureus play a crucial role in various cellular processes and host interactions. Consisting of a protein and a lipid moiety, they support nutrient acquisition and anchor the protein to the bacterial membrane. Recently, we identified several processed and secreted small linear peptides that derive from the secretion signal sequence of S. aureus lipoproteins. Here, we show, for the first time, that the protein moiety of the S. aureus lipoprotein CamS has a biological role that is distinct from its associated linear peptide staph-cAM373. The small peptide was shown to be involved in interspecies horizontal gene transfer, the primary mechanism for the dissemination of antibiotic resistance among bacteria. We provide evidence that the CamS protein moiety is a potent repressor of cytotoxins, such as α-toxin and leukocidins. The CamS-mediated suppression of toxin transcription was reflected by altered disease severity in in vivo infection models involving skin and soft tissue, as well as bloodstream infections. Collectively, we have uncovered the role of the protein moiety of the staphylococcal lipoprotein CamS as a previously uncharacterized repressor of S. aureus toxin production, which consequently regulates virulence and disease outcomes. Notably, the camS gene is conserved in S. aureus, and we also demonstrated the muted transcriptional response of cytotoxins in 2 different S. aureus lineages. Our findings provide the first evidence of distinct biological functions of the protein moiety and its associated linear peptide for a specific lipoprotein. Therefore, lipoproteins in S. aureus consist of 3 functional components: a lipid moiety, a protein moiety, and a small linear peptide, with putative different biological roles that might not only determine the outcome of host-pathogen interactions but also drive the acquisition of antibiotic resistance determinants.

Reality check: how do we know what's real?

How do we know what is real and what is merely a figment of our imagination? Dijkstra and Fleming tackle this question in a recent study. In contrast to the classic Perky effect, they found that once imagery crosses a 'reality threshold', it becomes difficult to distinguish from reality.

Multisensory subtypes of aphantasia: Mental imagery as supramodal perception in reverse.

Cognitive neuroscience research on mental imagery has largely focused on the visual imagery modality in unimodal task contexts. Recent studies have uncovered striking individual differences in visual imagery capacity, with some individuals reporting a subjective absence of conscious visual imagery ability altogether ("aphantasia"). However, naturalistic mental imagery is often multi-sensory, and preliminary findings suggest that many individuals with aphantasia also report a subjective lack of mental imagery in other sensory domains (such as auditory or olfactory imagery). In this paper, we perform a series of cluster analyses on the multi-sensory imagery questionnaire scores of two large groups of aphantasic subjects, defining latent sub-groups in this sample population. We demonstrate that aphantasia is a heterogenous phenomenon characterised by dominant sub-groups of individuals with visual aphantasia (those who report selective visual imagery absence) and multi-sensory aphantasia (those who report an inability to generate conscious mental imagery in any sensory modality). We replicate our findings in a second large sample and show that more unique aphantasia sub-types also exist, such as individuals with selectively preserved mental imagery in only one sensory modality (e.g. intact auditory imagery). We outline the implications of our findings for network theories of mental imagery, discussing how unique aphantasia aetiologies with distinct self-report patterns might reveal alterations to various levels of the sensory processing hierarchy implicated in mental imagery.

The impact of nutritional immunity on Group B streptococcal pathogenesis during wound infection.

Group B Streptococcus (GBS) is a Gram-positive pathobiont that can cause adverse health outcomes in neonates and vulnerable adult populations. GBS is one of the most frequently isolated bacteria from diabetic (Db) wound infections but is rarely found in the non-diabetic (nDb) wound environment. Previously, RNA sequencing of wound tissue from Db wound infections in leprdb diabetic mice showed increased expression of neutrophil factors, and genes involved in GBS metal transport such as the zinc (Zn), manganese (Mn), and putative nickel (Ni) import systems. Here, we develop a Streptozotocin-induced diabetic wound model to evaluate the pathogenesis of two invasive strains of GBS, serotypes Ia and V. We observe an increase in metal chelators such as calprotectin (CP) and lipocalin-2 during diabetic wound infections compared to nDb. We find that CP limits GBS survival in wounds of non-diabetic mice but does not impact survival in diabetic wounds. Additionally, we utilize GBS metal transporter mutants and determine that the Zn, Mn, and putative Ni transporters in GBS are dispensable in diabetic wound infection but contributed to bacterial persistence in non-diabetic animals. Collectively, these data suggest that in non-diabetic mice, functional nutritional immunity mediated by CP is effective at mitigating GBS infection, whereas in diabetic mice, the presence of CP is not sufficient to control GBS wound persistence. IMPORTANCE Diabetic wound infections are difficult to treat and often become chronic due to an impaired immune response as well as the presence of bacterial species that establish persistent infections. Group B Streptococcus (GBS) is one of the most frequently isolated bacterial species in diabetic wound infections and, as a result, is one of the leading causes of death from skin and subcutaneous infection. However, GBS is notoriously absent in non-diabetic wounds, and little is known about why this species thrives in diabetic infection. The work herein investigates how alterations in diabetic host immunity may contribute to GBS success during diabetic wound infection.

Group B Streptococcus adaptation promotes survival in a hyperinflammatory diabetic wound environment.

Diabetic wounds have poor healing outcomes due to the presence of numerous pathogens and a dysregulated immune response. Group B Streptococcus (GBS) is commonly isolated from diabetic wound infections, but the mechanisms of GBS virulence during these infections have not been investigated. Here, we develop a murine model of GBS diabetic wound infection and, using dual RNA sequencing, demonstrate that GBS infection triggers an inflammatory response. GBS adapts to this hyperinflammatory environment by up-regulating virulence factors including those known to be regulated by the two-component system covRS, such as the surface protein pbsP, and the cyl operon, which is responsible for hemolysin/pigmentation production. We recover hyperpigmented/hemolytic GBS colonies from the murine diabetic wound, which we determined encode mutations in covR. We further demonstrate that GBS mutants in cylE and pbsP are attenuated in the diabetic wound. This foundational study provides insight into the pathogenesis of GBS diabetic wound infections.

The Small RNA Teg41 Is a Pleiotropic Regulator of Virulence in Staphylococcus aureus.

Previously, our group demonstrated a role for the small RNA (sRNA) Teg41 in regulating production of the alpha phenol-soluble modulin toxins (αPSMs) in Staphylococcus aureus. Overexpressing Teg41 increased αPSM production while deleting the 3' end of Teg41 (Teg41Δ3' strain) resulted in a decrease in αPSM production, reduced hemolytic activity of S. aureus culture supernatants, and attenuated virulence in a murine abscess model of infection. In this study, we further explore the attenuation of virulence in the Teg41Δ3' strain. Using both localized and systemic models of infection, we demonstrate that the Teg41Δ3' strain is more severely attenuated than an ΔαPSM mutant, strongly suggesting that Teg41 influences more than the αPSMs. Proteomic and transcriptomic analysis of the wild-type and Teg41Δ3' strains reveals widespread alterations in transcript abundance and protein production in the absence of Teg41, confirming that Teg41 has pleiotropic effects in the cell. We go on to investigate the molecular mechanism underlying Teg41-mediated gene regulation. Surprisingly, results demonstrate that certain Teg41 target genes, including the αPSMs and ßPSMs, are transcriptionally altered in the Teg41Δ3' strain, while other targets, specifically spa (encoding surface protein A), are regulated at the level of transcript stability. Collectively, these data demonstrate that Teg41 is a pleiotropic RNA regulator in S. aureus that influences expression of a variety of genes using multiple different mechanisms.

Genomic Analyses Identify Manganese Homeostasis as a Driver of Group B Streptococcal Vaginal Colonization.

Group B Streptococcus (GBS) is associated with severe infections in utero and in newborn populations, including pneumonia, sepsis, and meningitis. GBS vaginal colonization of the pregnant mother is an important prerequisite for transmission to the newborn and the development of neonatal invasive disease; however, our understanding of the factors required for GBS persistence and ascension in the female reproductive tract (FRT) remains limited. Here, we utilized a GBS mariner transposon (Krmit) mutant library previously developed by our group and identified underrepresented mutations in 535 genes that contribute to survival within the vaginal lumen and colonization of vaginal, cervical, and uterine tissues. From these mutants, we identified 47 genes that were underrepresented in all samples collected, including mtsA, a component of the mtsABC locus, encoding a putative manganese (Mn2+)-dependent ATP-binding cassette transporter. RNA sequencing analysis of GBS recovered from the vaginal tract also revealed a robust increase of mtsA expression during vaginal colonization. We engineered an ΔmtsA mutant strain and found by using inductively coupled plasma mass spectrometry that it exhibited decreased concentrations of intracellular Mn2+, confirming its involvement in Mn2+ acquisition. The ΔmtsA mutant was significantly more susceptible to the metal chelator calprotectin and to oxidative stressors, including both H2O2 and paraquat, than wild-type (WT) GBS. We further observed that the ΔmtsA mutant strain exhibited a significant fitness defect in comparison to WT GBS in vivo by using a murine model of vaginal colonization. Taken together, these data suggest that Mn2+ homeostasis is an important process contributing to GBS survival in the FRT. IMPORTANCE Morbidity and mortality associated with GBS begin with colonization of the female reproductive tract (FRT). To date, our understanding of the factors required for GBS persistence in this environment remain limited. We identified several necessary systems for initial colonization of the vaginal lumen and penetration into the reproductive tissues via transposon mutagenesis sequencing. We determined that mutations in mtsA, the gene encoding a protein putatively involved in manganese (Mn2+) transport, were significantly underrepresented in all in vivo samples collected. We also show that mtsA contributes to Mn2+ acquisition and GBS survival during metal limitation by calprotectin, a metal-chelating protein complex. We further demonstrate that a mutant lacking mtsA is hypersusceptible to oxidative stress induced by both H2O2 and paraquat and has a severe fitness defect compared to WT GBS in the murine vaginal tract. This work reveals the importance of Mn2+ homeostasis at the host-pathogen interface in the FRT.

Memories with a blind mind: Remembering the past and imagining the future with aphantasia.

Our capacity to re-experience the past and simulate the future is thought to depend heavily on visual imagery, which allows us to construct complex sensory representations in the absence of sensory stimulation. There are large individual differences in visual imagery ability, but their impact on autobiographical memory and future prospection remains poorly understood. Research in this field assumes the normative use of visual imagery as a cognitive tool to simulate the past and future, however some individuals lack the ability to visualise altogether (a condition termed "aphantasia"). Aphantasia represents a rare and naturally occurring knock-out model for examining the role of visual imagery in episodic memory recall. Here, we assessed individuals with aphantasia on an adapted form of the Autobiographical Interview, a behavioural measure of the specificity and richness of episodic details underpinning the memory of events. Aphantasic participants generated significantly fewer episodic details than controls for both past and future events. This effect was most pronounced for novel future events, driven by selective reductions in visual detail retrieval, accompanied by comparatively reduced ratings of the phenomenological richness of simulated events, and paralleled by quantitative linguistic markers of reduced perceptual language use in aphantasic participants compared to those with visual imagery. Our findings represent the first systematic evidence (using combined objective and subjective data streams) that aphantasia is associated with a diminished ability to re-experience the past and simulate the future, indicating that visual imagery is an important cognitive tool for the dynamic retrieval and recombination of episodic details during mental simulation.

The novel protein ScrA acts through the SaeRS two-component system to regulate virulence gene expression in Staphylococcus aureus.

Staphylococcus aureus is a Gram-positive commensal that can also cause a variety of infections in humans. S. aureus virulence factor gene expression is under tight control by a complex regulatory network, which includes, sigma factors, sRNAs, and two-component systems (TCS). Previous work in our laboratory demonstrated that overexpression of the sRNA tsr37 leads to an increase in bacterial aggregation. Here, we demonstrate that the clumping phenotype is dependent on a previously unannotated 88 amino acid protein encoded within the tsr37 sRNA transcript (which we named ScrA for S. aureus clumping regulator A). To investigate the mechanism of action of ScrA we performed proteomics and transcriptomics in a ScrA overexpressing strain and show that a number of surface adhesins are upregulated, while secreted proteases are downregulated. Results also showed upregulation of the SaeRS TCS, suggesting that ScrA is influencing SaeRS activity. Overexpression of ScrA in a saeR mutant abrogates the clumping phenotype confirming that ScrA functions via the Sae system. Finally, we identified the ArlRS TCS as a positive regulator of scrA expression. Collectively, our results show that ScrA is an activator of the SaeRS system and suggests that ScrA may act as an intermediary between the ArlRS and SaeRS systems.

The pupillary light response as a physiological index of aphantasia, sensory and phenomenological imagery strength.

The pupillary light response is an important automatic physiological response which optimizes light reaching the retina. Recent work has shown that the pupil also adjusts in response to illusory brightness and a range of cognitive functions, however, it remains unclear what exactly drives these endogenous changes. Here, we show that the imagery pupillary light response correlates with objective measures of sensory imagery strength. Further, the trial-by-trial phenomenological vividness of visual imagery is tracked by the imagery pupillary light response. We also demonstrated that a group of individuals without visual imagery (aphantasia) do not show any significant evidence of an imagery pupillary light response, however they do show perceptual pupil light responses and pupil dilation with larger cognitive load. Our results provide evidence that the pupillary light response indexes the sensory strength of visual imagery. This work also provides the first physiological validation of aphantasia.

Global Annotation, Expression Analysis, and Stability of Candidate sRNAs in Group B Streptococcus.

Small, noncoding RNAs (sRNAs) are being increasingly identified as important regulatory molecules in prokaryotes. Due to the prevalence of next-generation sequencing-based techniques, such as RNA sequencing (RNA-seq), there is potential for increased discovery of sRNAs within bacterial genomes; however, these elements are rarely included in annotation files. Consequently, expression values for sRNAs are omitted from most transcriptomic analyses, and mechanistic studies have lagged behind those of protein regulators in numerous bacteria. Two previous studies have identified sRNAs in the human pathogen group B Streptococcus (GBS). Here, we utilize the data from these studies to create updated genome annotation files for the model GBS strains NEM316 and COH1. Using the updated COH1 annotation file, we reanalyze publicly available GBS RNA-seq whole-transcriptome data from GenBank to monitor GBS sRNA expression under a variety of conditions and genetic backgrounds. This analysis generated expression values for 232 putative sRNAs that were overlooked in previous transcriptomic analyses in 21 unique comparisons. To demonstrate the utility of these data, we identify an sRNA that is upregulated during vaginal colonization and demonstrate that overexpression of this sRNA leads to increased bacterial invasion into host epithelial cells. Finally, to monitor RNA degradation, we perform a transcript stability assay to identify highly stable sRNAs and compare stability profiles of sRNA- and protein-coding genes. Collectively, these data provide a wealth of transcriptomic data for putative sRNAs in GBS and a platform for future mechanistic studies. IMPORTANCE In recent years, sRNAs have emerged as potent regulatory molecules in bacteria, including numerous streptococcal species, and contribute to diverse processes, including stress response, metabolism, housekeeping, and virulence regulation. Improvements in sequencing technologies and in silico analyses have facilitated identification of these regulatory molecules as well as improved attempts to determine the location of sRNA genes on the genome. However, despite these advancements, sRNAs are rarely included in genome annotation files. Consequently, these molecules are often omitted from transcriptomic data analyses and are commonly repeat identified across multiple studies. Updating current genomes to include sRNA genes is therefore critical for better understanding bacterial regulation.

Temperature Influences the Composition and Cytotoxicity of Extracellular Vesicles in Staphylococcus aureus.

Staphylococcus aureus is a pathogenic bacterium but also a commensal of skin and anterior nares in humans. As S. aureus transits from skins/nares to inside the human body, it experiences changes in temperature. The production and content of S. aureus extracellular vesicles (EVs) have been increasingly studied over the past few years, and EVs are increasingly being recognized as important to the infectious process. Nonetheless, the impact of temperature variation on S. aureus EVs has not been studied in detail, as most reports that investigate EV cargoes and host cell interactions are performed using vesicles produced at 37°C. Here, we report that EVs in S. aureus differ in size and protein/RNA cargo depending on the growth temperature used. We demonstrate that the temperature-dependent regulation of vesicle production in S. aureus is mediated by the alpha phenol-soluble modulin peptides (αPSMs). Through proteomic analysis, we observed increased packaging of virulence factors at 40°C, whereas the EV proteome has greater diversity at 34°C. Similar to the protein content, we perform transcriptomic analysis and demonstrate that the RNA cargo also is impacted by temperature. Finally, we demonstrate greater αPSM- and alpha-toxin-mediated erythrocyte lysis with 40°C EVs, but 34°C EVs are more cytotoxic toward THP-1 cells. Together, our study demonstrates that small temperature variations have great impact on EV biogenesis and shape the interaction with host cells. IMPORTANCE Extracellular vesicles (EVs) are lipid bilayer spheres that contain proteins, nucleic acids, and lipids secreted by bacteria. They are involved in Staphylococcus aureus infections, as they package virulence factors and deliver their contents inside host cells. The impact of temperature variations experienced by S. aureus during the infectious process on EVs is unknown. Here, we demonstrate the importance of temperature in vesicle production and packaging. High temperatures promote packaging of virulence factors and increase the protein and lipid concentration but reduce the overall RNA abundance and protein diversity in EVs. The importance of temperature changes is highlighted by the fact that EVs produced at low temperature are more toxic toward macrophages, whereas EVs produced at high temperature display more hemolysis toward erythrocytes. Our research brings new insights into temperature-dependent vesiculation and interaction with the host during S. aureus transition from colonization to virulence.

Visual working memory in aphantasia: Retained accuracy and capacity with a different strategy.

Visual working memory paradigms involve retaining and manipulating visual information in mind over a period of seconds. Evidence suggests that visual imagery (sensory recruitment) is a strategy used by many to retain visual information during such tasks, leading some researchers to propose that visual imagery and visual working memory may be one and the same. If visual imagery is essential to visual working memory task performance there should be large ramifications for a special population of individuals who do not experience visual imagery, aphantasia. Here we assessed visual working memory task performance in this population using a number of different lab and clinical working memory tasks. We found no differences in capacity limits for visual, general number or spatial working memory for aphantasic individuals compared to controls. Further, aphantasic individuals showed no significant differences in performance on visual components of clinical working memory tests as compared to verbal components. However, there were significant differences in the reported strategies used by aphantasic individuals across all memory tasks. Additionally, aphantasic individual's visual memory accuracy did not demonstrate a significant oblique orientation effect, which is proposed to occur due to sensory recruitment, further supporting their non-visual imagery strategy reports. Taken together these data demonstrate that aphantasic individuals are not impaired on visual working memory tasks, suggesting visual imagery and working memory are not one and the same, with imagery (and sensory recruitment) being just one of the tools that can be used to solve visual working memory tasks.

Detection and Quantification of Secreted Nuclease Activity in Staphylococcus aureus Culture Supernatants.

Staphylococcal secreted nuclease contributes to S. aureus virulence by degrading neutrophil extracellular traps (NETs), which allows the bacterium to evade the host immune system and has also been shown to promote biofilm dispersal. In this chapter, two methods for detecting nuclease activity are described, both of which have increased sensitivity compared to the traditional nuclease agar method.