Most free-living organisms require the synthesis and/or acquisition of purines and pyrimidines, which form the basis of nucleotides, to survive. In most bacteria, the nucleotides are synthesized de ...novo and the products are used in many cell functions, including DNA replication, energy storage, and as signaling molecules. Due to their central role in the metabolism of bacteria, both nucleotide biosynthesis pathways have strong links with the virulence of opportunistic and bona fide bacterial pathogens. Recent findings have established a new, shared link in the control of nucleotide biosynthesis and the production of virulence factors. Furthermore, targeting of these pathways forms the basis of interspecies competition and can provide an open source for new antimicrobial compounds. Here, we highlight the contribution of nucleotide biosynthesis to bacterial pathogenesis in a plethora of different diseases and speculate on how they can be targeted by intervention strategies.
De novo nucleotide biosynthesis is essential for the survival and virulence of pathogenic bacterial species in vitro and in vivo.Regulators of nucleotide biosynthesis are emerging as important for control of the expression of virulence factors.De novo nucleotide biosynthesis is targeted by purpose-specific molecules to facilitate bacterial survival during interspecies competition.Targeting of nucleotide biosynthesis can act as an antivirulence strategy, either on its own or in conjunction with existing antimicrobial therapies.
is a noted human and animal pathogen. Despite decades of research on this important bacterium, there are still many unanswered questions regarding the pathogenic mechanisms it uses to infect the ...mammalian host. This can be attributed to it possessing a plethora of virulence factors and complex virulence factor and metabolic regulation. PurR, the purine biosynthesis regulator, was recently also shown to regulate virulence factors in
, and mutations in
result in derepression of fibronectin binding proteins (FnBPs) and extracellular toxins, required for a so-called hypervirulent phenotype. Here, we show that hypervirulent strains containing
mutations can be attenuated with the addition of purine biosynthesis mutations, implicating the necessity for
purine biosynthesis in this phenotype and indicating that
in the mammalian host experiences purine limitation. Using cell culture, we showed that while
mutants are not altered in epithelial cell binding, compared to that of wild-type (WT)
,
mutants have enhanced invasion of these nonprofessional phagocytes, consistent with the requirement of FnBPs for invasion of these cells. This correlates with
mutants having increased transcription of
genes, resulting in higher levels of surface-exposed FnBPs to promote invasion. These data provide important contributions to our understanding of how the pathogenesis of
is affected by sensing of purine levels during infection of the mammalian host.
Coagulase-negative staphylococci and Staphylococcus aureus colonize similar niches in mammals and conceivably compete for space and nutrients. Here, we report that a coagulase-negative ...staphylococcus, Staphylococcus chromogenes ATCC43764, synthesizes and secretes 6-thioguanine (6-TG), a purine analog that suppresses S. aureus growth by inhibiting de novo purine biosynthesis. We identify a 6-TG biosynthetic gene cluster in S. chromogenes and other coagulase-negative staphylococci including S. epidermidis, S. pseudintermedius and S. capitis. Recombinant S. aureus strains harbouring this operon produce 6-TG and, when used in subcutaneous co-infections in mice with virulent S. aureus USA300, protect the host from necrotic lesion formation. Used prophylactically, 6-TG reduces necrotic skin lesions in mice infected with USA300, and this effect is mediated by abrogation of toxin production. RNAseq analyses reveal that 6-TG downregulates expression of genes coding for purine biosynthesis, the accessory gene regulator (agr) and ribosomal proteins in S. aureus, providing an explanation for its effect on toxin production.
Staphylococcus aureus is a significant cause of human infection. Here, we demonstrate that mutations in the transcriptional repressor of purine biosynthesis, purR, enhance the pathogenic potential of ...S. aureus. Indeed, systemic infection with purR mutants causes accelerated mortality in mice, which is due to aberrant up-regulation of fibronectin binding proteins (FnBPs). Remarkably, purR mutations can arise upon exposure of S. aureus to stress, such as an intact immune system. In humans, naturally occurring anti-FnBP antibodies exist that, while not protective against recurrent S. aureus infection, ostensibly protect against hypervirulent S. aureus infections. Vaccination studies support this notion, where anti-Fnb antibodies in mice protect against purR hypervirulence. These findings provide a novel link between purine metabolism and virulence in S. aureus.
We recently discovered that 6-thioguanine (6-TG) is an antivirulence compound that is produced by a number of coagulase-negative staphylococci. In Staphylococcus aureus, it inhibits
purine ...biosynthesis and ribosomal protein expression, thus inhibiting growth and abrogating toxin production. Mechanisms by which S. aureus may develop resistance to this compound are currently unknown. Here, we show that 6-TG-resistant S. aureus mutants emerge spontaneously when the bacteria are subjected to high concentrations of 6-TG
. Whole-genome sequencing of these mutants revealed frameshift and missense mutations in a xanthine-uracil permease family protein (
ix
hio
uanine
ermease) and single nucleotide polymorphisms in hypoxanthine phosphoribosyltransferase (
). These mutations engender S. aureus the ability to resist both the growth inhibitory and toxin downregulation effects of 6-TG. While prophylactic administration of 6-TG ameliorates necrotic lesions in subcutaneous infection of mice with methicillin-resistant S. aureus (MRSA) strain USA300 LAC, the drug did not reduce lesion size formed by the 6-TG-resistant strains. These findings identify mechanisms of 6-TG resistance, and this information can be leveraged to inform strategies to slow the evolution of resistance.
is a foremost bacterial pathogen responsible for a vast array of human diseases. Staphylococcal superantigens (SAgs) constitute a family of exotoxins from
that bind directly to major ...histocompatibility complex (MHC) class II and T cell receptors to drive extensive T cell activation and cytokine release. Although these toxins have been implicated in serious disease, including toxic shock syndrome, the specific pathological mechanisms remain unclear. Herein, we aimed to elucidate how SAgs contribute to pathogenesis during bloodstream infections and utilized transgenic mice encoding human MHC class II to render mice susceptible to SAg activity. We demonstrate that SAgs contribute to
bacteremia by massively increasing bacterial burden in the liver, and this was mediated by CD4
T cells that produced interferon gamma (IFN-γ) to high levels in a SAg-dependent manner. Bacterial burdens were reduced by blocking IFN-γ, phenocopying SAg-deletion mutant strains, and inhibiting a proinflammatory response. Infection kinetics and flow cytometry analyses suggested that this was a macrophage-driven mechanism, which was confirmed through macrophage-depletion experiments. Experiments in human cells demonstrated that excessive IFN-γ allowed
to replicate efficiently within macrophages. This indicates that SAgs promote bacterial survival by manipulating the immune response to inhibit effective clearing of
Altogether, this work implicates SAg toxins as critical therapeutic targets for preventing persistent or severe
disease.
is the leading cause of pyoderma in dogs and is often associated with recurrent skin infections that require prolonged antibiotic therapy. High levels of antibiotic use have led to multidrug ...resistance, including the emergence of epidemic methicillin-resistant clones. Our understanding of the pathogenesis of
skin infection is very limited, and the identification of the key host-pathogen interactions underpinning infection could lead to the design of novel therapeutic or vaccine-based approaches for controlling disease. Here, we employ a novel murine cutaneous-infection model of
and investigate the role of the two cell wall-associated proteins (SpsD and SpsL) in skin disease pathogenesis. Experimental infection with wild-type
strain ED99 or a gene-deletion derivative deficient in expression of SpsD led to a focal accumulation of neutrophils and necrotic debris in the dermis and deeper tissues of the skin characteristic of a classical cutaneous abscess. In contrast, mice infected with mutants deficient in SpsL or both SpsD and SpsL developed larger cutaneous lesions with distinct histopathological features of regionally extensive cellulitis rather than focal abscessation. Furthermore, comparison of the bacterial loads in
induced cutaneous lesions revealed a significantly increased burden of bacteria in the mice infected with SpsL-deficient mutants. These findings reveal a key role for SpsL in murine skin abscess formation and highlight a novel function for a bacterial surface protein in determining the clinical outcome and pathology of infection caused by a major canine pathogen.
Bacterial superantigens (SAgs) cause Vβ-dependent T-cell proliferation leading to immune dysregulation associated with the pathogenesis of life-threatening infections such as toxic shock syndrome, ...and necrotizing pneumonia. Previously, we demonstrated that staphylococcal enterotoxin-like toxin X (SElX) from Staphylococcus aureus is a classical superantigen that exhibits T-cell activation in a Vβ-specific manner, and contributes to the pathogenesis of necrotizing pneumonia. Here, we discovered that SElX can also bind to neutrophils from human and other mammalian species and disrupt IgG-mediated phagocytosis. Site-directed mutagenesis of the conserved sialic acid-binding motif of SElX abolished neutrophil binding and phagocytic killing, and revealed multiple glycosylated neutrophil receptors for SElX binding. Furthermore, the neutrophil binding-deficient mutant of SElX retained its capacity for T-cell activation demonstrating that SElX exhibits mechanistically independent activities on distinct cell populations associated with acquired and innate immunity, respectively. Finally, we demonstrated that the neutrophil-binding activity rather than superantigenicity is responsible for the SElX-dependent virulence observed in a necrotizing pneumonia rabbit model of infection. Taken together, we report the first example of a SAg, that can manipulate both the innate and adaptive arms of the human immune system during S. aureus pathogenesis.
Bacterial co-infection is one of the most common complications of SARS CoV-2 infection. Here, we present a protocol for the in vitro study of co-infection between SARS CoV-2 and Staphylococcus ...aureus. We describe steps for quantifying viral and bacterial replication kinetics in the same sample, with the optional extraction of host RNA and proteins. This protocol is applicable to many viral and bacterial strains and can be performed in different cell types.
For complete details on the use and execution of this protocol, please refer to Goncheva et al.1
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•In vitro technique to study co-infection between SARS CoV-2 and Staphylococcus aureus•Simultaneous quantification of viral and bacterial replication•Steps to perform parallel measurements of host RNA and/or protein•Compatible with different viral and bacterial strains
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Bacterial co-infection is one of the most common complications of SARS CoV-2 infection. Here, we present a protocol for the in vitro study of co-infection between SARS CoV-2 and Staphylococcus aureus. We describe steps for quantifying viral and bacterial replication kinetics in the same sample, with the optional extraction of host RNA and proteins. This protocol is applicable to many viral and bacterial strains and can be performed in different cell types.
Influenza A virus (IAV) causes annual epidemics of respiratory disease in humans, often complicated by secondary coinfection with bacterial pathogens such as
Here, we report that the
secreted protein ...lipase 1 enhances IAV replication
in primary cells, including human lung fibroblasts. The proviral activity of lipase 1 is dependent on its enzymatic function, acts late in the viral life cycle, and results in increased infectivity through positive modulation of virus budding. Furthermore, the proviral effect of lipase 1 on IAV is exhibited during
infection of embryonated hen's eggs and, importantly, increases the yield of a vaccine strain of IAV by approximately 5-fold. Thus, we have identified the first
protein to enhance IAV replication, suggesting a potential role in coinfection. Importantly, this activity may be harnessed to address global shortages of influenza vaccines.
Influenza A virus (IAV) causes annual epidemics and sporadic pandemics of respiratory disease. Secondary bacterial coinfection by organisms such as
is the most common complication of primary IAV infection and is associated with high levels of morbidity and mortality. Here, we report the first identified
factor (lipase 1) that enhances IAV replication during infection via positive modulation of virus budding. The effect is observed
in embryonated hen's eggs and greatly enhances the yield of a vaccine strain, a finding that could be applied to address global shortages of influenza vaccines.
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