Manganese uptake by MtsABC contributes to the pathogenesis of human pathogen group A streptococcus by resisting host nutritional immune defenses.

The interplay between host nutritional immune mechanisms and bacterial nutrient uptake systems has a major impact on the disease outcome. The host immune factor calprotectin (CP) limits the availability of essential transition metals, such as manganese (Mn) and zinc (Zn), to control the growth of invading pathogens. We previously demonstrated that the competition between CP and the human pathogen group A streptococcus (GAS) for Zn impacts GAS pathogenesis. However, the contribution of Mn sequestration by CP in GAS infection control and the role of GAS Mn acquisition systems in overcoming host-imposed Mn limitation remain unknown. Using a combination of in vitro and in vivo studies, we show that GAS-encoded mtsABC is a Mn uptake system that aids bacterial evasion of CP-imposed Mn scarcity and promotes GAS virulence. Mn deficiency caused by either the inactivation of mtsC or CP also impaired the protective function of GAS-encoded Mn-dependent superoxide dismutase. Our ex vivo studies using human saliva show that saliva is a Mn-scant body fluid, and Mn acquisition by MtsABC is critical for GAS survival in human saliva. Finally, animal infection studies using wild-type (WT) and CP-/- mice showed that MtsABC is critical for GAS virulence in WT mice but dispensable in mice lacking CP, indicating the direct interplay between MtsABC and CP in vivo. Together, our studies elucidate the role of the Mn import system in GAS evasion of host-imposed metal sequestration and underscore the translational potential of MtsABC as a therapeutic or prophylactic target.

Spatio-temporal dynamics of intra-host variability in SARS-CoV-2 genomes.

During the course of the COVID-19 pandemic, large-scale genome sequencing of SARS-CoV-2 has been useful in tracking its spread and in identifying variants of concern (VOC). Viral and host factors could contribute to variability within a host that can be captured in next-generation sequencing reads as intra-host single nucleotide variations (iSNVs). Analysing 1347 samples collected till June 2020, we recorded 16 410 iSNV sites throughout the SARS-CoV-2 genome. We found ∼42% of the iSNV sites to be reported as SNVs by 30 September 2020 in consensus sequences submitted to GISAID, which increased to ∼80% by 30th June 2021. Following this, analysis of another set of 1774 samples sequenced in India between November 2020 and May 2021 revealed that majority of the Delta (B.1.617.2) and Kappa (B.1.617.1) lineage-defining variations appeared as iSNVs before getting fixed in the population. Besides, mutations in RdRp as well as RNA-editing by APOBEC and ADAR deaminases seem to contribute to the differential prevalence of iSNVs in hosts. We also observe hyper-variability at functionally critical residues in Spike protein that could alter the antigenicity and may contribute to immune escape. Thus, tracking and functional annotation of iSNVs in ongoing genome surveillance programs could be important for early identification of potential variants of concern and actionable interventions.

Skeletal muscle mitochondrial dysfunction mediated by Pseudomonas aeruginosa quorum-sensing transcription factor MvfR: reversing effects with anti-MvfR and mitochondrial-targeted compounds.

Sepsis and chronic infections with Pseudomonas aeruginosa, a leading "ESKAPE" bacterial pathogen, are associated with increased morbidity and mortality and skeletal muscle atrophy. The actions of this pathogen on skeletal muscle remain poorly understood. In skeletal muscle, mitochondria serve as a crucial energy source, which may be perturbed by infection. Here, using the well-established backburn and infection model of murine P. aeruginosa infection, we deciphered the systemic impact of the quorum-sensing transcription factor MvfR (multiple virulence factor regulator) by interrogating, 5 days post-infection, its effect on mitochondrial-related functions in the gastrocnemius skeletal muscle and the outcome of the pharmacological inhibition of MvfR function and that of the mitochondrial-targeted peptide, Szeto-Schiller 31 (SS-31). Our findings show that the MvfR perturbs adenosine triphosphate generation, oxidative phosphorylation, and antioxidant response, elevates the production of reactive oxygen species, and promotes oxidative damage of mitochondrial DNA in the gastrocnemius muscle of infected mice. These impairments in mitochondrial-related functions were corroborated by the alteration of key mitochondrial proteins involved in electron transport, mitochondrial biogenesis, dynamics and quality control, and mitochondrial uncoupling. Pharmacological inhibition of MvfR using the potent anti-MvfR lead, D88, we developed, or the mitochondrial-targeted peptide SS-31 rescued the MvfR-mediated alterations observed in mice infected with the wild-type strain PA14. Our study provides insights into the actions of MvfR in orchestrating mitochondrial dysfunction in the skeletal murine muscle, and it presents novel therapeutic approaches for optimizing clinical outcomes in affected patients. IMPORTANCE: Skeletal muscle, pivotal for many functions in the human body, including breathing and protecting internal organs, contains abundant mitochondria essential for maintaining cellular homeostasis during infection. The effect of Pseudomonas aeruginosa (PA) infections on skeletal muscle remains poorly understood. Our study delves into the role of a central quorum-sensing transcription factor, multiple virulence factor regulator (MvfR), that controls the expression of multiple acute and chronic virulence functions that contribute to the pathogenicity of PA. The significance of our study lies in the role of MvfR in the metabolic perturbances linked to mitochondrial functions in skeletal muscle and the effectiveness of the novel MvfR inhibitor and the mitochondrial-targeted peptide SS-31 in alleviating the mitochondrial disturbances caused by PA in skeletal muscle. Inhibiting MvfR or interfering with its effects can be a potential therapeutic strategy to curb PA virulence.

Skeletal Muscle Mitochondrial Dysfunction Mediated by Pseudomonas aeruginosa Quorum Sensing Transcription Factor MvfR: Reversing Effects with Anti-MvfR and Mitochondrial-Targeted Compounds.

Sepsis and chronic infections with Pseudomonas aeruginosa, a leading "ESKAPE" bacterial pathogen, are associated with increased morbidity and mortality and skeletal muscle atrophy. The actions of this pathogen on skeletal muscle remain poorly understood. In skeletal muscle, mitochondria serve as a crucial energy source, which may be perturbed by infection. Here, using the well-established backburn and infection model of murine P. aeruginosa infection, we deciphered the systemic impact of the quorum sensing (QS) transcription factor MvfR by interrogating five days post-infection its effect on mitochondrial-related functions in the gastrocnemius skeletal muscle and the outcome of the pharmacological inhibition of MvfR function and that of the mitochondrial-targeted peptide, Szeto-Schiller 31 (SS-31). Our findings show that the MvfR perturbs ATP generation, oxidative phosphorylation (OXPHOS), and antioxidant response, elevates the production of reactive oxygen species, and promotes oxidative damage of mitochondrial DNA in the gastrocnemius muscle of infected mice. These impairments in mitochondrial-related functions were corroborated by the alteration of key mitochondrial proteins involved in electron transport, mitochondrial biogenesis, dynamics and quality control, and mitochondrial uncoupling. Pharmacological inhibition of MvfR using the potent anti-MvfR lead, D88, we developed, or the mitochondrial-targeted peptide SS-31 rescued the MvfR- mediated alterations observed in mice infected with the wild-type strain PA14. Our study provides insights into the actions of MvfR in orchestrating mitochondrial dysfunction in the skeletal murine muscle, and it presents novel therapeutic approaches for optimizing clinical outcomes in affected patients.

Engineered probiotic overcomes pathogen defences using signal interference and antibiotic production to treat infection in mice.

Probiotic supplements are suggested to promote human health by preventing pathogen colonization. However, the mechanistic bases for their efficacy in vivo are largely uncharacterized. Here using metabolomics and bacterial genetics, we show that the human oral probiotic Streptococcus salivarius K12 (SAL) produces salivabactin, an antibiotic that effectively inhibits pathogenic Streptococcus pyogenes (GAS) in vitro and in mice. However, prophylactic dosing with SAL enhanced GAS colonization in mice and ex vivo in human saliva. We showed that, on co-colonization, GAS responds to a SAL intercellular peptide signal that controls SAL salivabactin production. GAS produces a secreted protease, SpeB, that targets SAL-derived salivaricins and enhances GAS survival. Using this knowledge, we re-engineered probiotic SAL to prevent signal eavesdropping by GAS and potentiate SAL antimicrobials. This engineered probiotic demonstrated superior efficacy in preventing GAS colonization in vivo. Our findings show that knowledge of interspecies interactions can identify antibiotic- and probiotic-based strategies to combat infection.

Attenuation of quorum sensing system and virulence in Vibrio cholerae by phytomolecules.

The Vibrio cholerae, a gram-negative bacterium, is the causative agent of cholera. Quorum sensing is a cell-to-cell communication that leads to gene expression, accumulation of signaling molecules, biofilm formation, and production of virulence factors. The quorum sensing pathway in V. cholerae is regulated by luxO, and biofilm formation and other virulence factors are positively controlled by aphA and negatively by hapR. Hence, targeting the global regulator luxO would be a promising approach to modulate the QS to curtail V. cholerae pathogenesis. The present study investigated the modulating activity of quercetin and naringenin on biofilm formation and quorum-sensing regulated phenotypes in V. cholerae. Then after we determined the anti-quorum sensing capability of phytomolecules against the model organism Chromobacterium violaceum. Also, we performed flow cytometry for live/dead bacteria, MTT assay, CLSM, and growth curve analysis to determine their role as QS modulators rather than anti-bacterial. V. cholerae strains VC287 and N16961 formed thick biofilm. We observed a two-fold reduction in the expression of biofilm-associated genes comprising gbpA, vpsA, rbmA, and mbaA in the presence of phytomolecules indicating that phytomolecules modulate quorum sensing pathway rather than killing the bacteria. These phytomolecules were non-toxic and non-hemolytic and had anti-adhesion and anti-invasion properties. In addition, quercetin and naringenin were found to be highly effective compared to known quorum-sensing inhibitors terrein and furanone C-30. Thus, this study provides evidence that phytomolecules: quercetin and naringenin modulate the quorum-sensing pathway rather than killing the bacteria and can be used as an anti-quorum-sensing molecule for therapy against the pathogen.

The leaderless communication peptide (LCP) class of quorum-sensing peptides is broadly distributed among Firmicutes.

The human pathogen Streptococcus pyogenes secretes a short peptide (leaderless communication peptide, LCP) that mediates intercellular communication and controls bacterial virulence through interaction with its receptor, RopB. Here, we show that LCP and RopB homologues are present in other Firmicutes. We experimentally validate that LCPs with distinct peptide communication codes act as bacterial intercellular signals and regulate gene expression in Streptococcus salivarius, Streptococcus porcinus, Enterococcus malodoratus and Limosilactobacillus reuteri. Our results indicate that LCPs are more widespread than previously thought, and their characterization may uncover new signaling mechanisms and roles in coordinating diverse bacterial traits.

Managing Manganese: The Role of Manganese Homeostasis in Streptococcal Pathogenesis.

Pathogenic streptococci require manganese for survival in the host. In response to invading pathogens, the host recruits nutritional immune effectors at infection sites to withhold manganese from the pathogens and control bacterial growth. The manganese scarcity impairs several streptococcal processes including oxidative stress defenses, de novo DNA synthesis, bacterial survival, and virulence. Emerging evidence suggests that pathogens also encounter manganese toxicity during infection and manganese excess impacts streptococcal virulence by manganese mismetallation of non-cognate molecular targets involved in bacterial antioxidant defenses and cell division. To counter host-imposed manganese stress, the streptococcal species employ a sophisticated sensory system that tightly coordinates manganese stress-specific molecular strategies to negate host induced manganese stress and proliferate in the host. Here we review the molecular details of host-streptococcal interactions in the battle for manganese during infection and the significance of streptococcal effectors involved to bacterial pathophysiology.

Long-read 16S-seq reveals nasopharynx microbial dysbiosis and enrichment of Mycobacterium and Mycoplasma in COVID-19 patients: a potential source of co-infection.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major global health concern. This virus infects the upper respiratory tract and causes pneumonia-like symptoms. So far, few studies have shown alterations in nasopharyngeal (NP) microbial diversity, enrichment of opportunistic pathogens and their role in co-infections during respiratory infections. Therefore, we hypothesized that microbial diversity changes, with increase in the population of opportunistic pathogens, during SARS-CoV2 infection in the nasopharynx, which may be involved in co-infection in COVID-19 patients. The 16S rRNA variable regions, V1-V9, of NP samples of control and COVID-19 (symptomatic and asymptomatic) patients were sequenced using the Oxford Nanopore™ technology. Comprehensive bioinformatics analysis for determining alpha/beta diversities, non-metric multidimensional scaling, correlation studies, canonical correspondence analysis, linear discriminate analysis, and dysbiosis index were used to analyze the control and COVID-19-specific NP microbiomes. We observed significant dysbiosis in the COVID-19 NP microbiome with an increase in the abundance of opportunistic pathogens at genus and species levels in asymptomatic/symptomatic patients. The significant abundance of Mycobacteria spp. and Mycoplasma spp. in symptomatic patients suggests their association and role in co-infections in COVID-19 patients. Furthermore, we found strong correlation of enrichment of Mycobacteria and Mycoplasma with the occurrences of chest pain and fever in symptomatic COVID-19 patients. This is the first study from India to show the abundance of Mycobacteria and Mycoplasma opportunistic pathogens in non-hospitalized COVID-19 patients and their relationship with symptoms, indicating the possibility of co-infections.

Extracellular vesicles: An emerging platform in gram-positive bacteria.

Extracellular vesicles (EV), also known as membrane vesicles, are produced as an end product of secretion by both pathogenic and non-pathogenic bacteria. Several reports suggest that archaea, gram-negative bacteria, and eukaryotic cells secrete membrane vesicles as a means for cell-free intercellular communication. EVs influence intercellular communication by transferring a myriad of biomolecules including genetic information. Also, EVs have been implicated in many phenomena such as stress response, intercellular competition, lateral gene transfer, and pathogenicity. However, the cellular process of secreting EVs in gram-positive bacteria is less studied. A notion with the thick cell-walled microbes such as gram-positive bacteria is that the EV release is impossible among them. The role of gram-positive EVs in health and diseases is being studied gradually. Being nano-sized, the EVs from gram-positive bacteria carry a diversity of cargo compounds that have a role in bacterial competition, survival, invasion, host immune evasion, and infection. In this review, we summarise the current understanding of the EVs produced by gram-positive bacteria. Also, we discuss the functional aspects of these components while comparing them with gram-negative bacteria.

Antibiotic Susceptibility, Virulence Pattern, and Typing of Staphylococcus aureus Strains Isolated From Variety of Infections in India.

Staphylococcus aureus is one of the major causes of nosocomial infections. This organism produces powerful toxins and cause superficial lesions, systemic infections, and several toxemic syndromes. A total of 109 S. aureus strains isolated from a variety of infections like ocular diseases, wound infection, and sputum were included in the study. Minimum inhibitory concentration (MIC) was determined against 8 antimicrobials. PCR determined the presence of 16S rRNA, nuc, mecA, czrC, qacA/B, pvl, and toxin genes in S. aureus isolates. Pulse-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), SCCmec, spa-, and agr-typing and serotyping determined the diversity among them. All isolates of S. aureus were resistant to two or more than two antibiotics and generated 32 resistance patterns. These isolates were positive for 16S rRNA and S. aureus-specific nuc gene, but showed variable results for mecA, czrC, and qacA/B and pvl genes. Of the 32 methicillin-resistant S. aureus (MRSA), 13 strains carried SCCmec type V, seven type IV, two type III, and nine carried unreported type UT6. Of the 109 strains, 98.2% were positive for hlg, 94.5% for hla, 86.2% for sei, 73.3% for efb, 70.6% for cna, 30.2% for sea, and 12.8% for sec genes. Serotypes VII and VI were prevalent among S. aureus strains. PFGE analysis grouped the 109 strains into 77 clusters. MLST classified the strains into 33 sequence types (ST) and eight clonal complexes (CCs) of which 12 were singletons, and two belong to new allelic profiles. Isolates showed 46 spa-types that included two new spa-types designated as t14911 and t14912. MRSA and methicillin-susceptible S. aureus (MSSA) isolates were diverse in terms of antibiotic resistance pattern, toxin genotypes, SCCmec types, serotypes and PFGE, MLST, and spa-types. However, few isolates from eye infection and wound infection belong to CC239, ST239, and spa-type t037/t657. The study thus suggests that S. aureus strains are multidrug resistant, virulent, and diverse irrespective of sources and place of isolation. These findings necessitate the continuous surveillance of multidrug-resistant and virulent S. aureus and monitoring of the transmission of infection.