Isolation and Characterization of New Bacteriophages against Staphylococcal Clinical Isolates from Diabetic Foot Ulcers.

Staphylococcus sp. is the most common bacterial genus in infections related to diabetic foot ulcers (DFUs). The emergence of multidrug-resistant bacteria places a serious burden on public health systems. Phage therapy is an alternative treatment to antibiotics, overcoming the issue of antibiotic resistance. In this study, six phages (SAVM01 to SAVM06) were isolated from effluents and were used against a panel of staphylococcal clinical samples isolated from DFUs. A genomic analysis revealed that the phages belonged to the Herelleviridae family, with sequences similar to those of the Kayvirus genus. No lysogeny-associated genes, known virulence or drug resistance genes were identified in the phage genomes. The phages displayed a strong lytic and antibiofilm activity against DFU clinical isolates, as well as against opportunistic pathogenic coagulase-negative staphylococci. The results presented here suggest that these phages could be effective biocontrol agents against staphylococcal clinical isolates from DFUs.

The secreted tyrosine phosphatase PtpA promotes Staphylococcus aureus survival in RAW 264.7 macrophages through decrease of the SUMOylation host response.

IMPORTANCE: Staphylococcus aureus uses numerous strategies to survive and persist in the intracellular environment of professional phagocytes, including modulation of the SUMOylation process. This study aims to understand how S. aureus alters host SUMOylation to enhance its intracellular survival in professional phagocytes. Our results indicate that S. aureus strain Newman utilizes PtpA-driven phosphorylation to decrease the amount of SUMOylated proteins in murine macrophages to facilitate its survival in this immune cell type.

Druggable redox pathways against Mycobacterium abscessus in cystic fibrosis patient-derived airway organoids.

Mycobacterium abscessus (Mabs) drives life-shortening mortality in cystic fibrosis (CF) patients, primarily because of its resistance to chemotherapeutic agents. To date, our knowledge on the host and bacterial determinants driving Mabs pathology in CF patient lung remains rudimentary. Here, we used human airway organoids (AOs) microinjected with smooth (S) or rough (R-)Mabs to evaluate bacteria fitness, host responses to infection, and new treatment efficacy. We show that S Mabs formed biofilm, and R Mabs formed cord serpentines and displayed a higher virulence. While Mabs infection triggers enhanced oxidative stress, pharmacological activation of antioxidant pathways resulted in better control of Mabs growth and reduced virulence. Genetic and pharmacological inhibition of the CFTR is associated with better growth and higher virulence of S and R Mabs. Finally, pharmacological activation of antioxidant pathways inhibited Mabs growth, at least in part through the quinone oxidoreductase NQO1, and improved efficacy in combination with cefoxitin, a first line antibiotic. In conclusion, we have established AOs as a suitable human system to decipher mechanisms of CF-driven respiratory infection by Mabs and propose boosting of the NRF2-NQO1 axis as a potential host-directed strategy to improve Mabs infection control.

The ROSA-Like Prophage Colonizing Staphylococcus aureus Promotes Intracellular Survival, Biofilm Formation, and Virulence in a Chronic Wound Environment.

BACKGROUND: The transition from colonization to invasion is critical in diabetic foot ulcer (DFU). Staphylococcus aureus can colonize DFU, or invade the underlying tissues, causing serious infections. The ROSA-like prophage has previously been implicated in strain colonization characteristics of S aureus isolates in uninfected ulcers. METHODS: In this study, we investigated this prophage in the S aureus-colonizing strain using an in vitro chronic wound medium mimicking the chronic wound environment. RESULTS: Chronic wound medium reduced bacterial growth and increased biofilm formation and virulence in a zebrafish model. CONCLUSIONS: The ROSA-like prophage promoted intracellular survival of S aureus-colonizing strain in macrophages, keratinocytes, and osteoblasts.

Phosphorylation-mediated regulation of the Bacillus anthracis phosphoglycerate mutase by the Ser/Thr protein kinase PrkC.

Bacillus anthracis Ser/Thr protein kinase PrkC is necessary for phenotypic memory and spore germination, and the loss of PrkC-dependent phosphorylation events affect the spore development. During sporulation, Bacillus sp. can store 3-Phosphoglycerate (3-PGA) that will be required at the onset of germination when ATP will be necessary. The Phosphoglycerate mutase (Pgm) catalyzes the isomerization of 2-PGA and 3-PGA and is important for spore germination as a key metabolic enzyme that maintains 3-PGA pool at later events. Therefore, regulation of Pgm is important for an efficient spore germination process and metabolic switching. While the increased expression of Pgm in B. anthracis decreases spore germination efficiency, it remains unexplored if PrkC could directly influence Pgm activity. Here, we report the phosphorylation and regulation of Pgm by PrkC and its impact on Pgm stability and catalytic activity. Mass spectrometry revealed Pgm phosphorylation on seven threonine residues. In silico mutational analysis highlighted the role of Thr459 residue towards metal and substrate binding. Altogether, we demonstrated that PrkC-mediated Pgm phosphorylation negatively regulates its activity that is essential to maintain Pgm in its apo-like isoform before germination. This study advances the role of Pgm regulation that represents an important switch for B. anthracis resumption of metabolism and spore germination.

Phenotypic and Genotypic Virulence Characterisation of Staphylococcus pettenkoferi Strains Isolated from Human Bloodstream and Diabetic Foot Infections.

Staphylococcus pettenkoferi is a recently described coagulase-negative Staphylococcus identified in human diseases, especially in infections of foot ulcers in patients living with diabetes mellitus. To date, its pathogenicity remains underexplored. In this study, whole-genome analysis was performed on a collection of 29 S. pettenkoferi clinical strains isolated from bloodstream and diabetic foot infections with regard to their phylogenetic relationships and comprehensive analysis of their resistome and virulome. Their virulence was explored by their ability to form biofilm, their growth kinetics and in an in vivo zebrafish embryo infection model. Our results identified two distinct clades (I and II) and two subclades (I-a and I-b) with notable genomic differences. All strains had a slow bacterial growth. Three profiles of biofilm formation were noted, with 89.7% of isolates able to produce biofilm and harbouring a high content of biofilm-encoding genes. Two virulence profiles were also observed in the zebrafish model irrespective of the strains' origin or biofilm profile. Therefore, this study brings new insights in S. pettenkoferi pathogenicity.

Characterization of the Secreted Acid Phosphatase SapS Reveals a Novel Virulence Factor of Staphylococcus aureus That Contributes to Survival and Virulence in Mice.

Staphylococcus aureus possesses a large arsenal of immune-modulating factors, enabling it to bypass the immune system's response. Here, we demonstrate that the acid phosphatase SapS is secreted during macrophage infection and promotes its intracellular survival in this type of immune cell. In animal models, the SA564 sapS mutant demonstrated a significantly lower bacterial burden in liver and renal tissues of mice at four days post infection in comparison to the wild type, along with lower pathogenicity in a zebrafish infection model. The SA564 sapS mutant elicits a lower inflammatory response in mice than the wild-type strain, while S. aureus cells harbouring a functional sapS induce a chemokine response that favours the recruitment of neutrophils to the infection site. Our in vitro and quantitative transcript analysis show that SapS has an effect on S. aureus capacity to adapt to oxidative stress during growth. SapS is also involved in S. aureus biofilm formation. Thus, this study shows for the first time that SapS plays a significant role during infection, most likely through inhibiting a variety of the host's defence mechanisms.

Bacterial Interactions in the Context of Chronic Wound Biofilm: A Review.

Chronic wounds, defined by their resistance to care after four weeks, are a major concern, affecting millions of patients every year. They can be divided into three types of lesions: diabetic foot ulcers (DFU), pressure ulcers (PU), and venous/arterial ulcers. Once established, the classical treatment for chronic wounds includes tissue debridement at regular intervals to decrease biofilm mass constituted by microorganisms physiologically colonizing the wound. This particular niche hosts a dynamic bacterial population constituting the bed of interaction between the various microorganisms. The temporal reshuffle of biofilm relies on an organized architecture. Microbial community turnover is mainly associated with debridement (allowing transitioning from one major representant to another), but also with microbial competition and/or collaboration within wounds. This complex network of species and interactions has the potential, through diversity in antagonist and/or synergistic crosstalk, to accelerate, delay, or worsen wound healing. Understanding these interactions between microorganisms encountered in this clinical situation is essential to improve the management of chronic wounds.

Bacteriophage Therapy for Staphylococcus Aureus Infections: A Review of Animal Models, Treatments, and Clinical Trials.

Staphylococcus aureus (S. aureus) is a common and virulent human pathogen causing several serious illnesses including skin abscesses, wound infections, endocarditis, osteomyelitis, pneumonia, and toxic shock syndrome. Antibiotics were first introduced in the 1940s, leading to the belief that bacterial illnesses would be eradicated. However, microorganisms, including S. aureus, began to develop antibiotic resistance from the increased use and abuse of antibiotics. Antibiotic resistance is now one of the most serious threats to global public health. Bacteria like methicillin-resistant Staphylococcus aureus (MRSA) remain a major problem despite several efforts to find new antibiotics. New treatment approaches are required, with bacteriophage treatment, a non-antibiotic strategy to treat bacterial infections, showing particular promise. The ability of S. aureus to resist a wide range of antibiotics makes it an ideal candidate for phage therapy studies. Bacteriophages have a relatively restricted range of action, enabling them to target pathogenic bacteria. Their usage, usually in the form of a cocktail of bacteriophages, allows for more focused treatment while also overcoming the emergence of resistance. However, many obstacles remain, particularly in terms of their effects in vivo, necessitating the development of animal models to assess the bacteriophage efficiency. Here, we provide a review of the animal models, the various clinical case treatments, and clinical trials for S. aureus phage therapy.

Random nature of epithelial cancer cell monolayers.

Although the polygonal shape of epithelial cells has been drawing the attention of scientists for several centuries, only a decade and a half ago it was demonstrated that distributions of polygon types (DOPTs) are similar in proliferative epithelia of many different plant and animal species. In this study, we show that hyper-proliferation of cancer cells disrupts this universal paradigm and results in randomly organized epithelial structures. Examining non-synchronized and synchronized HeLa cervix cells, we suppose that the spread of cell sizes is the main parameter controlling the DOPT in the cancer cell monolayers. To test this hypothesis, we develop a theory of morphologically similar random polygonal packings. By analysing differences between tumoural and normal epithelial cell monolayers, we conclude that the latter have more ordered structures because of their lower proliferation rates and, consequently, more effective relaxation of mechanical stress associated with cell division and growth. To explain the structural features of normal proliferative epithelium, we take into account the spread of cell sizes in the monolayer. The proposed theory also rationalizes some highly ordered unconventional post-mitotic epithelia.

The Transcription Factor SpoVG Is of Major Importance for Biofilm Formation of Staphylococcus epidermidis under In Vitro Conditions, but Dispensable for In Vivo Biofilm Formation.

Staphylococcus epidermidis is a common cause of device related infections on which pathogens form biofilms (i.e., multilayered cell populations embedded in an extracellular matrix). Here, we report that the transcription factor SpoVG is essential for the capacity of S. epidermidis to form such biofilms on artificial surfaces under in vitro conditions. Inactivation of spoVG in the polysaccharide intercellular adhesin (PIA) producing S. epidermidis strain 1457 yielded a mutant that, unlike its parental strain, failed to produce a clear biofilm in a microtiter plate-based static biofilm assay. A decreased biofilm formation capacity was also observed when 1457 ΔspoVG cells were co-cultured with polyurethane-based peripheral venous catheter fragments under dynamic conditions, while the cis-complemented 1457 ΔspoVG::spoVG derivative formed biofilms comparable to the levels seen with the wild-type. Transcriptional studies demonstrated that the deletion of spoVG significantly altered the expression of the intercellular adhesion (ica) locus by upregulating the transcription of the ica operon repressor icaR and down-regulating the transcription of icaADBC. Electrophoretic mobility shift assays (EMSA) revealed an interaction between SpoVG and the icaA-icaR intergenic region, suggesting SpoVG to promote biofilm formation of S. epidermidis by modulating ica expression. However, when mice were challenged with the 1457 ΔspoVG mutant in a foreign body infection model, only marginal differences in biomasses produced on the infected catheter fragments between the mutant and the parental strain were observed. These findings suggest that SpoVG is critical for the PIA-dependent biofilm formation of S. epidermis under in vitro conditions, but is largely dispensable for biofilm formation of this skin commensal under in vivo conditions.

Investigating Pathogenicity and Virulence of Staphylococcus pettenkoferi: An Emerging Pathogen.

Staphylococcus pettenkoferi is a coagulase-negative Staphylococcus identified in 2002 that has been implicated in human diseases as an opportunistic pathogenic bacterium. Its multiresistant character is becoming a major health problem, yet the pathogenicity of S. pettenkoferi is poorly characterized. In this study, the pathogenicity of a S. pettenkoferi clinical isolate from diabetic foot osteomyelitis was compared with a Staphylococcus aureus strain in various in vitro and in vivo experiments. Growth kinetics were compared against S. aureus, and bacteria survival was assessed in the RAW 264.7 murine macrophage cell line, the THP-1 human leukemia monocytic cell line, and the HaCaT human keratinocyte cell line. Ex vivo analysis was performed in whole blood survival assays and in vivo assays via the infection model of zebrafish embryos. Moreover, whole-genome analysis was performed. Our results show that S. pettenkoferi was able to survive in human blood, human keratinocytes, murine macrophages, and human macrophages. S. pettenkoferi demonstrated its virulence by causing substantial embryo mortality in the zebrafish model. Genomic analysis revealed virulence factors such as biofilm-encoding genes (e.g., icaABCD; rsbUVW) and regulator-encoding genes (e.g., agr, mgrA, sarA, saeS) well characterized in S. aureus. This study thus advances the knowledge of this under-investigated pathogen and validates the zebrafish infection model for this bacterium.

Staphylococcus aureus Toxins: An Update on Their Pathogenic Properties and Potential Treatments.

Staphylococcus aureus is a clinically important pathogen that causes a wide range of human infections, from minor skin infections to severe tissue infection and sepsis. S. aureus has a high level of antibiotic resistance and is a common cause of infections in hospitals and the community. The rising prevalence of community-acquired methicillin-resistant S. aureus (CA-MRSA), combined with the important severity of S. aureus infections in general, has resulted in the frequent use of anti-staphylococcal antibiotics, leading to increasing resistance rates. Antibiotic-resistant S. aureus continues to be a major health concern, necessitating the development of novel therapeutic strategies. S. aureus uses a wide range of virulence factors, such as toxins, to develop an infection in the host. Recently, anti-virulence treatments that directly or indirectly neutralize S. aureus toxins have showed promise. In this review, we provide an update on toxin pathogenic characteristics, as well as anti-toxin therapeutical strategies.

Long-Term Intrahost Evolution of Staphylococcus aureus Among Diabetic Patients With Foot Infections.

Staphylococcus aureus is one of the main pathogens isolated from diabetic foot infections (DFI). The purpose of this study was to evaluate the importance of the persistence of S. aureus in this environment and the possible modifications of the bacterial genome content over time. Molecular typing of S. aureus isolates cultured from patients with the same DFI over a 7-year study revealed a 25% rate of persistence of this species in 48 patients, with a short median persistence time of 12weeks (range: 4-52weeks). Non-specific clonal complexes were linked to this persistence. During the follow-up, bla genes were acquired in three cases, whereas some virulence markers were lost in all cases after a long period of colonization (21.5weeks). Only one patient (2%) had a long-term persistence of 48weeks. The genome sequencing of a clonal pair of early/late strains isolated in this patient showed mutations in genes encoding bacterial defence and two-component signal transduction systems. Although, this study suggests that the long-term persistence of S. aureus in DFI is a rare event, genomic evolution is observed, highlighting the low adaptive ability of S. aureus to the specific environment and stressful conditions of diabetic foot ulcers. These results provide the basis for better understanding of S. aureus dynamics during persistent colonization in chronic wounds.

Staphylococcus aureus Decreases SUMOylation Host Response to Promote Intramacrophage Survival.

Staphylococcus aureus is a commensal bacterium that causes severe infections in soft tissue and the bloodstream. During infection, S. aureus manipulates host cell response to facilitate its own replication and dissemination. Here, we show that S. aureus significantly decreases the level of SUMOylation, an essential post-translational modification, in infected macrophages 24 h post-phagocytosis. The reduced level of SUMOylation correlates with a decrease in the SUMO-conjugating enzyme Ubc9. The over-expression of SUMO proteins in macrophages impaired bacterial intracellular proliferation and the inhibition of SUMOylation with ML-792 increased it. Together, these findings demonstrated for the first time the role of host SUMOylation response toward S. aureus infection.

The Low-Molecular Weight Protein Arginine Phosphatase PtpB Affects Nuclease Production, Cell Wall Integrity, and Uptake Rates of Staphylococcus aureus by Polymorphonuclear Leukocytes.

The epidemiological success of Staphylococcus aureus as a versatile pathogen in mammals is largely attributed to its virulence factor repertoire and the sophisticated regulatory network controlling this virulon. Here we demonstrate that the low-molecular-weight protein arginine phosphatase PtpB contributes to this regulatory network by affecting the growth phase-dependent transcription of the virulence factor encoding genes/operons aur, nuc, and psmα, and that of the small regulatory RNA RNAIII. Inactivation of ptpB in S. aureus SA564 also significantly decreased the capacity of the mutant to degrade extracellular DNA, to hydrolyze proteins in the extracellular milieu, and to withstand Triton X-100 induced autolysis. SA564 ΔptpB mutant cells were additionally ingested faster by polymorphonuclear leukocytes in a whole blood phagocytosis assay, suggesting that PtpB contributes by several ways positively to the ability of S. aureus to evade host innate immunity.

The Phosphoarginine Phosphatase PtpB from Staphylococcus aureus Is Involved in Bacterial Stress Adaptation during Infection.

Staphylococcus aureus continues to be a public health threat, especially in hospital settings. Studies aimed at deciphering the molecular and cellular mechanisms that underlie pathogenesis, host adaptation, and virulence are required to develop effective treatment strategies. Numerous host-pathogen interactions were found to be dependent on phosphatases-mediated regulation. This study focused on the analysis of the role of the low-molecular weight phosphatase PtpB, in particular, during infection. Deletion of ptpB in S. aureus strain SA564 significantly reduced the capacity of the mutant to withstand intracellular killing by THP-1 macrophages. When injected into normoglycemic C57BL/6 mice, the SA564 ΔptpB mutant displayed markedly reduced bacterial loads in liver and kidney tissues in a murine S. aureus abscess model when compared to the wild type. We also observed that PtpB phosphatase-activity was sensitive to oxidative stress. Our quantitative transcript analyses revealed that PtpB affects the transcription of various genes involved in oxidative stress adaptation and infectivity. Thus, this study disclosed first insights into the physiological role of PtpB during host interaction allowing us to link phosphatase-dependent regulation to oxidative bacterial stress adaptation during infection.

Dictyostelium lacking the single atlastin homolog Sey1 shows aberrant ER architecture, proteolytic processes and expansion of the Legionella-containing vacuole.

Dictyostelium discoideum Sey1 is the single ortholog of mammalian atlastin 1-3 (ATL1-3), which are large homodimeric GTPases mediating homotypic fusion of endoplasmic reticulum (ER) tubules. In this study, we generated a D. discoideum mutant strain lacking the sey1 gene and found that amoebae deleted for sey1 are enlarged, but grow and develop similarly to the parental strain. The ∆sey1 mutant amoebae showed an altered ER architecture, and the tubular ER network was partially disrupted without any major consequences for other organelles or the architecture of the secretory and endocytic pathways. Macropinocytic and phagocytic functions were preserved; however, the mutant amoebae exhibited cumulative defects in lysosomal enzymes exocytosis, intracellular proteolysis, and cell motility, resulting in impaired growth on bacterial lawns. Moreover, ∆sey1 mutant cells showed a constitutive activation of the unfolded protein response pathway (UPR), but they still readily adapted to moderate levels of ER stress, while unable to cope with prolonged stress. In D. discoideum ∆sey1 the formation of the ER-associated compartment harbouring the bacterial pathogen Legionella pneumophila was also impaired. In the mutant amoebae, the ER was less efficiently recruited to the "Legionella-containing vacuole" (LCV), the expansion of the pathogen vacuole was inhibited at early stages of infection and intracellular bacterial growth was reduced. In summary, our study establishes a role of D. discoideum Sey1 in ER architecture, proteolysis, cell motility and intracellular replication of L. pneumophila.

Methylation of two-component response regulator MtrA in mycobacteria negatively modulates its DNA binding and transcriptional activation.

Post-translational modifications such as phosphorylation, nitrosylation, and pupylation modulate multiple cellular processes in Mycobacterium tuberculosis. While protein methylation at lysine and arginine residues is widespread in eukaryotes, to date only two methylated proteins in Mtb have been identified. Here, we report the identification of methylation at lysine and/or arginine residues in nine mycobacterial proteins. Among the proteins identified, we chose MtrA, an essential response regulator of a two-component signaling system, which gets methylated on multiple lysine and arginine residues to examine the functional consequences of methylation. While methylation of K207 confers a marginal decrease in the DNA-binding ability of MtrA, methylation of R122 or K204 significantly reduces the interaction with the DNA. Overexpression of S-adenosyl homocysteine hydrolase (SahH), an enzyme that modulates the levels of S-adenosyl methionine in mycobacteria decreases the extent of MtrA methylation. Most importantly, we show that decreased MtrA methylation results in transcriptional activation of mtrA and sahH promoters. Collectively, we identify novel methylated proteins, expand the list of modifications in mycobacteria by adding arginine methylation, and show that methylation regulates MtrA activity. We propose that protein methylation could be a more prevalent modification in mycobacterial proteins.

The protein kinase PknB negatively regulates biosynthesis and trafficking of mycolic acids in mycobacteria.

Mycobacterium tuberculosis is the causative agent of tuberculosis and remains one of the most widespread and deadliest bacterial pathogens in the world. A distinguishing feature of mycobacteria that sets them apart from other bacteria is the unique architecture of their cell wall, characterized by various species-specific lipids, most notably mycolic acids (MAs). Therefore, targeted inhibition of enzymes involved in MA biosynthesis, transport, and assembly has been extensively explored in drug discovery. Additionally, more recent evidence suggests that many enzymes in the MA biosynthesis pathway are regulated by kinase-mediated phosphorylation, thus opening additional drug-development opportunities. However, how phosphorylation regulates MA production remains unclear. Here, we used genetic strategies combined with lipidomics and phosphoproteomics approaches to investigate the role of protein phosphorylation in Mycobacterium The results of this analysis revealed that the Ser/Thr protein kinase PknB regulates the export of MAs and promotes the remodeling of the mycobacterial cell envelope. In particular, we identified the essential MmpL3 as a substrate negatively regulated by PknB. Taken together, our findings add to the understanding of how PknB activity affects the mycobacterial MA biosynthesis pathway and reveal the essential role of protein phosphorylation/dephosphorylation in governing lipid metabolism, paving the way for novel antimycobacterial strategies.