Inflammation and Neutrophil Oxidative Burst in a Family with NFKB1 p.R157X LOF and Sterile Necrotizing Fasciitis.

Loss-of-function (LOF) mutations in NFKB1, coding for p105, may cause common variable immunodeficiency due to dysregulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κΒ) pathway. Monoallelic LOF variants of NFKB1 can predispose to uncontrolled inflammation including sterile necrotizing fasciitis or pyoderma gangrenosum. In this study, we explored the impact of a heterozygous NFKB1 c.C936T/p.R157X LOF variant on immunity in sterile fasciitis patients and their family members. The p50 or p105 protein levels were reduced in all variant carriers. Interleukin-1ß (IL-1ß) and interleukin-8 (IL-8) levels were elevated in vitro, potentially contributing to the very high neutrophil counts observed during fasciitis episodes. Phosphorylation of p65/RelA was reduced in p.R157X neutrophils suggesting defective activation of canonical NF-κB. Oxidative burst after NF-κB-independent phorbol 12-myristate 13-acetate (PMA) stimulation was similar in both p.R157X and control neutrophils. Comparable amounts of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex subunits were found in p.R157X and control neutrophils. However, a compromised oxidative burst was observed in p.R157X neutrophils following activation of NF-κB-dependent mechanisms following stimulation of toll-like receptor 2 (TLR2) and Dectin-1. Neutrophil extracellular trap formation was not affected by p.R157X. In summary, the NFKB1 c.C936T/p.R157X LOF variant has an impact on inflammation and neutrophil function and may play a role in the pathogenesis of sterile necrotizing fasciitis.

Atypical Presentation of Severe Fungal Necrotizing Fasciitis in a Patient with X-Linked Agammaglobulinemia.

X-linked agammaglobulinemia is a rare primary immunodeficiency due to a BTK mutation. The patients are characteristically deficient in peripheral B cells and serum immunoglobulins. While they are susceptible to infections caused by bacteria, enteroviruses, and parasites, fungal infections are uncommon in XLA patients. Here, we report a boy of Malay ethnicity who suffered from recurrent upper respiratory tract infections and severe progressive necrotizing fasciitis caused by Saksenaea erythrospora. Immunological tests showed a B cell deficiency and hypogammaglobulinemia. Whole-exome sequencing identified a dinucleotide deletion (c.1580_1581del) in BTK, confirmed by Sanger sequencing and predicted to be disease causing by in silico functional prediction tools (Varsome and MutationTaster2) but was absent in the gnomAD database. This mutation resulted in a frameshift and premature termination (p.C527fs), which disrupted the protein structure. The mother was heterozygous at the mutation site, confirming her carrier status. Flow cytometric analysis of monocyte BTK expression showed it to be absent in the patient and bimodal in the mother. This study describes a novel BTK mutation in a defined hotspot and an atypical fungal phenotype in XLA. Further studies are required to understand the pathogenesis of fungal infection in XLA.

Phenotype and multi-omics comparison of Staphylococcus and Streptococcus uncovers pathogenic traits and predicts zoonotic potential.

BACKGROUND: Staphylococcus and Streptococcus species can cause many different diseases, ranging from mild skin infections to life-threatening necrotizing fasciitis. Both genera consist of commensal species that colonize the skin and nose of humans and animals, and of which some can display a pathogenic phenotype. RESULTS: We compared 235 Staphylococcus and 315 Streptococcus genomes based on their protein domain content. We show the relationships between protein persistence and essentiality by integrating essentiality predictions from two metabolic models and essentiality measurements from six large-scale transposon mutagenesis experiments. We identified clusters of strains within species based on proteins associated to similar biological processes. We built Random Forest classifiers that predicted the zoonotic potential. Furthermore, we identified shared attributes between of Staphylococcus aureus and Streptococcus pyogenes that allow them to cause necrotizing fasciitis. CONCLUSIONS: Differences observed in clustering of strains based on functional groups of proteins correlate with phenotypes such as host tropism, capability to infect multiple hosts and drug resistance. Our method provides a solid basis towards large-scale prediction of phenotypes based on genomic information.

DNA methylation from a Type I restriction modification system influences gene expression and virulence in Streptococcus pyogenes.

DNA methylation is pervasive across all domains of life. In bacteria, the presence of N6-methyladenosine (m6A) has been detected among diverse species, yet the contribution of m6A to the regulation of gene expression is unclear in many organisms. Here we investigated the impact of DNA methylation on gene expression and virulence within the human pathogen Streptococcus pyogenes, or Group A Streptococcus. Single Molecule Real-Time sequencing and subsequent methylation analysis identified 412 putative m6A sites throughout the 1.8 Mb genome. Deletion of the Restriction, Specificity, and Methylation gene subunits (ΔRSM strain) of a putative Type I restriction modification system lost all detectable m6A at the recognition sites and failed to prevent transformation with foreign-methylated DNA. RNA-sequencing identified 20 genes out of 1,895 predicted coding regions with significantly different gene expression. All of the differentially expressed genes were down regulated in the ΔRSM strain relative to the parent strain. Importantly, we found that the presence of m6A DNA modifications affected expression of Mga, a master transcriptional regulator for multiple virulence genes, surface adhesins, and immune-evasion factors in S. pyogenes. Using a murine subcutaneous infection model, mice infected with the ΔRSM strain exhibited an enhanced host immune response with larger skin lesions and increased levels of pro-inflammatory cytokines compared to mice infected with the parent or complemented mutant strains, suggesting alterations in m6A methylation influence virulence. Further, we found that the ΔRSM strain showed poor survival within human neutrophils and reduced adherence to human epithelial cells. These results demonstrate that, in addition to restriction of foreign DNA, gram-positive bacteria also use restriction modification systems to regulate the expression of gene networks important for virulence.

Gene fitness landscape of group A streptococcus during necrotizing myositis.

Necrotizing fasciitis and myositis are devastating infections characterized by high mortality. Group A streptococcus (GAS) is a common cause of these infections, but the molecular pathogenesis is poorly understood. We report a genome-wide analysis using serotype M1 and M28 strains that identified GAS genes contributing to necrotizing myositis in nonhuman primates (NHP), a clinically relevant model. Using transposon-directed insertion-site sequencing (TraDIS), we identified 126 and 116 GAS genes required for infection by serotype M1 and M28 organisms, respectively. For both M1 and M28 strains, more than 25% of the GAS genes required for necrotizing myositis encode known or putative transporters. Thirteen GAS transporters contributed to both M1 and M28 strain fitness in NHP myositis, including putative importers for amino acids, carbohydrates, and vitamins and exporters for toxins, quorum-sensing peptides, and uncharacterized molecules. Targeted deletion of genes encoding 5 transporters confirmed that each isogenic mutant strain was significantly (P < 0.05) impaired in causing necrotizing myositis in NHPs. Quantitative reverse-transcriptase PCR (qRT-PCR) analysis showed that these 5 genes are expressed in infected NHP and human skeletal muscle. Certain substrate-binding lipoproteins of these transporters, such as Spy0271 and Spy1728, were previously documented to be surface exposed, suggesting that our findings have translational research implications.

Association between cytokine response, the LRINEC score and outcome in patients with necrotising soft tissue infection: a multicentre, prospective study.

Early assessment of necrotising soft tissue infection (NSTI) is challenging. Analysis of inflammatory markers could provide important information about disease severity and guide decision making. For this purpose, we investigated the association between cytokine levels and the Laboratory Risk Indicator for Necrotising Fasciitis (LRINEC)-score, disease severity and mortality in NSTI patients. In 159 patients, plasma was analysed for IL-1ß, IL-6, IL-10 and TNF-α upon admission. The severity of NSTI was assessed by SAPS, SOFA score, septic shock, microbial aetiology, renal replacement therapy and amputation. We found no significant difference in cytokine levels according to a LRINEC- score above or below 6 (IL-1ß: 3.0 vs. 1.3; IL-6: 607 vs. 289; IL-10: 38.4 vs. 38.8; TNF-α: 15.1 vs. 7.8 pg/mL, P > 0.05). Patients with ß-haemolytic streptococcal infection had higher level of particularly IL-6. There was no difference in mortality between patients with a LRINEC-score above or below 6. In the adjusted analysis assessing 30-day mortality, the association was strongest for IL-1ß (OR 3.86 [95% CI, 1.43-10.40], P = 0.008) and IL-10 (4.80 [1.67-13.78], P = 0.004). In conclusion, we found no significant association between the LRINEC-score and cytokine levels on admission. IL-6 was consistently associated with disease severity, whereas IL-1ß had the strongest association with 30-day mortality.

Genetic Architecture of Group A Streptococcal Necrotizing Soft Tissue Infections in the Mouse.

Host genetic variations play an important role in several pathogenic diseases, and we have previously provided strong evidences that these genetic variations contribute significantly to differences in susceptibility and clinical outcomes of invasive Group A Streptococcus (GAS) infections, including sepsis and necrotizing soft tissue infections (NSTIs). Our initial studies with conventional mouse strains revealed that host genetic variations and sex differences play an important role in orchestrating the severity, susceptibility and outcomes of NSTIs. To understand the complex genetic architecture of NSTIs, we utilized an unbiased, forward systems genetics approach in an advanced recombinant inbred (ARI) panel of mouse strains (BXD). Through this approach, we uncovered interactions between host genetics, and other non-genetic cofactors including sex, age and body weight in determining susceptibility to NSTIs. We mapped three NSTIs-associated phenotypic traits (i.e., survival, percent weight change, and lesion size) to underlying host genetic variations by using the WebQTL tool, and identified four NSTIs-associated quantitative genetic loci (QTL) for survival on mouse chromosome (Chr) 2, for weight change on Chr 7, and for lesion size on Chr 6 and 18 respectively. These QTL harbor several polymorphic genes. Identification of multiple QTL highlighted the complexity of the host-pathogen interactions involved in NSTI pathogenesis. We then analyzed and rank-ordered host candidate genes in these QTL by using the QTLminer tool and then developed a list of 375 candidate genes on the basis of annotation data and biological relevance to NSTIs. Further differential expression analyses revealed 125 genes to be significantly differentially regulated in susceptible strains compared to their uninfected controls. Several of these genes are involved in innate immunity, inflammatory response, cell growth, development and proliferation, and apoptosis. Additional network analyses using ingenuity pathway analysis (IPA) of these 125 genes revealed interleukin-1 beta network as key network involved in modulating the differential susceptibility to GAS NSTIs.

A molecular trigger for intercontinental epidemics of group A Streptococcus.

The identification of the molecular events responsible for strain emergence, enhanced virulence, and epidemicity has been a long-pursued goal in infectious diseases research. A recent analysis of 3,615 genomes of serotype M1 group A Streptococcus strains (the so-called "flesh-eating" bacterium) identified a recombination event that coincides with the global M1 pandemic beginning in the early 1980s. Here, we have shown that the allelic variation that results from this recombination event, which replaces the chromosomal region encoding secreted NADase and streptolysin O, is the key driver of increased toxin production and enhanced infection severity of the M1 pandemic strains. Using isoallelic mutant strains, we found that 3 polymorphisms in this toxin gene region increase resistance to killing by human polymorphonuclear leukocytes, increase bacterial proliferation, and increase virulence in animal models of pharyngitis and necrotizing fasciitis. Genome sequencing of an additional 1,125 streptococcal strains and virulence studies revealed that a highly similar recombinational replacement event underlies an ongoing intercontinental epidemic of serotype M89 group A Streptococcus infections. By identifying the molecular changes that enhance upper respiratory tract fitness, increased resistance to innate immunity, and increased tissue destruction, we describe a mechanism that underpins epidemic streptococcal infections, which have affected many millions of people.

Label-free proteomic analysis of environmental acidification-influenced Streptococcus pyogenes secretome reveals a novel acid-induced protein histidine triad protein A (HtpA) involved in necrotizing fasciitis.

Streptococcus pyogenes is responsible for various diseases. During infection, bacteria must adapt to adverse environments, such as the acidic environment. Acidic stimuli may stimulate S. pyogenes to invade into deeper tissue. However, how this acidic stimulus causes S. pyogenes to manipulate its secretome for facilitating invasion remains unclear. The dynamic label-free LC-MS/MS profiling identified 97 proteins, which are influenced by environmental acidification. Among these, 33 (34%) of the identified proteins were predicted to be extracellular proteins. Interestingly, classical secretory proteins comprise approximately 90% of protein abundance of the secretome in acidic condition at the stationary phase. One acid-induced secreted protein, HtpA, was selected to investigate its role in invasive infection. The mouse infected by the htpA deficient mutant showed lower virulence and smaller lesion area than the wild-type strain. The mutant strain was more efficiently cleared at infected skin than the wild-type strain. Besides, the relative phagocytosis resistance is lower in the mutant strain than in the wild-type strain. These data indicate that a novel acid-induced virulence factor, HtpA, which improves anti-phagocytosis ability for causing necrotizing fasciitis. Our investigation provides vital information for documenting the broad influences and mechanisms underlying the invasive behavior of S. pyogenes in an acidified environment. BIOLOGICAL SIGNIFICANCE: The acidified infected environment may facilitate S. pyogenes invasion from the mucosa to the deeper subepithelial tissue. The acid stimuli have been considered to affect the complex regulatory network of S. pyogenes for causing severe infections. Many of secreted virulence factors influenced by acidified environment may also play a crucial role in pathogenesis of invasive disease. To investigate temporal secretome changes under acidic environment, a comparative secretomics approach using label-free LC-MS/MS was undertaken to analyze the secretome in acidic and neutral conditions. The dynamic label-free LC-MS/MS profiling and secretome prediction were used in this study for mining acid-influenced secreted proteins. We identified 33 acid-influenced secreted proteins in this study. Among these proteins, a novel acid-induced virulence factor, HtpA, was demonstrated to improve anti-phagocytosis ability for causing necrotizing fasciitis. In addition, our study demonstrates the first evidence that acidic stimuli and growth-phase cues are crucial for classical protein secretion in S. pyogenes.

Evolutionary pathway to increased virulence and epidemic group A Streptococcus disease derived from 3,615 genome sequences.

We sequenced the genomes of 3,615 strains of serotype Emm protein 1 (M1) group A Streptococcus to unravel the nature and timing of molecular events contributing to the emergence, dissemination, and genetic diversification of an unusually virulent clone that now causes epidemic human infections worldwide. We discovered that the contemporary epidemic clone emerged in stepwise fashion from a precursor cell that first contained the phage encoding an extracellular DNase virulence factor (streptococcal DNase D2, SdaD2) and subsequently acquired the phage encoding the SpeA1 variant of the streptococcal pyrogenic exotoxin A superantigen. The SpeA2 toxin variant evolved from SpeA1 by a single-nucleotide change in the M1 progenitor strain before acquisition by horizontal gene transfer of a large chromosomal region encoding secreted toxins NAD(+)-glycohydrolase and streptolysin O. Acquisition of this 36-kb region in the early 1980s into just one cell containing the phage-encoded sdaD2 and speA2 genes was the final major molecular event preceding the emergence and rapid intercontinental spread of the contemporary epidemic clone. Thus, we resolve a decades-old controversy about the type and sequence of genomic alterations that produced this explosive epidemic. Analysis of comprehensive, population-based contemporary invasive strains from seven countries identified strong patterns of temporal population structure. Compared with a preepidemic reference strain, the contemporary clone is significantly more virulent in nonhuman primate models of pharyngitis and necrotizing fasciitis. A key finding is that the molecular evolutionary events transpiring in just one bacterial cell ultimately have produced millions of human infections worldwide.

'Stalled' periocular necrotising fasciitis: early effective treatment or host genetic determinants?

BACKGROUND: Necrotising fasciitis (NF) is a devastating disease with considerable mortality and morbidity, and early aggressive surgical debridement of devitalised necrotic tissues has traditionally been advocated. METHODS: We describe three patients who were referred from other units several weeks after developing periocular necrotising fasciitis; in all the three, the disease had been managed medically without surgical debridement, with apparent 'stalling' of the inflammatory process despite persistent necrotic periocular tissue. RESULTS: Following 'elective debridement' of the devitalised tissues and reconstruction with local flaps, all achieved a satisfactory aesthetic result. DISCUSSION: The role of host genetic determinants, polarised cytokine responses, and early, effective medical treatment in patients with atypical 'disease phenotypes' in NF are discussed.

Antibody orientation at bacterial surfaces is related to invasive infection.

Several of the most significant bacterial pathogens in humans, including Streptococcus pyogenes, express surface proteins that bind IgG antibodies via their fragment crystallizable (Fc) region, and the dogma is that this protects the bacteria against phagocytic killing in blood. However, analysis of samples from a patient with invasive S. pyogenes infection revealed dramatic differences in the presence and orientation of IgG antibodies at the surface of bacteria from different sites. In the throat, IgG was mostly bound to the bacterial surface via Fc, whereas in the blood IgG was mostly bound via fragment antigen-binding (Fab). In infected and necrotic tissue, the Fc-binding proteins were removed from the bacterial surface. Further investigation showed that efficient bacterial IgGFc-binding occurs only in IgG-poor environments, such as saliva. As a consequence, the bacteria are protected against phagocytic killing, whereas in blood plasma where the concentration of IgG is high, the antibodies preferentially bind via Fab, facilitating opsonization and bacterial killing. IgG-poor environments represent the natural habitat for IgGFc-binding bacteria, and IgGFc-binding proteins may have evolved to execute their function in such environments. The lack of protection in plasma also helps to explain why cases of severe invasive infections with IgGFc-binding bacteria are so rare compared with superficial and uncomplicated infections.

Decreased necrotizing fasciitis capacity caused by a single nucleotide mutation that alters a multiple gene virulence axis.

Single-nucleotide changes are the most common cause of natural genetic variation among members of the same species, but there is remarkably little information bearing on how they alter bacterial virulence. We recently discovered a single-nucleotide mutation in the group A Streptococcus genome that is epidemiologically associated with decreased human necrotizing fasciitis ("flesh-eating disease"). Working from this clinical observation, we find that wild-type mtsR function is required for group A Streptococcus to cause necrotizing fasciitis in mice and nonhuman primates. Expression microarray analysis revealed that mtsR inactivation results in overexpression of PrsA, a chaperonin involved in posttranslational maturation of SpeB, an extracellular cysteine protease. Isogenic mutant strains that overexpress prsA or lack speB had decreased secreted protease activity in vivo and recapitulated the necrotizing fasciitis-negative phenotype of the DeltamtsR mutant strain in mice and monkeys. mtsR inactivation results in increased PrsA expression, which in turn causes decreased SpeB secreted protease activity and reduced necrotizing fasciitis capacity. Thus, a naturally occurring single-nucleotide mutation dramatically alters virulence by dysregulating a multiple gene virulence axis. Our discovery has broad implications for the confluence of population genomics and molecular pathogenesis research.

CD46 transgenic mouse model of necrotizing fasciitis caused by Streptococcus pyogenes infection.

We developed a human CD46-expressing transgenic (Tg) mouse model of subcutaneous (s.c.) infection into both hind footpads with clinically isolated 11 group A streptococcus (GAS) serotype M1 strains. When the severity levels of foot lesions at 72 h and the mortality rates by 336 h were compared after s.c. infection with 1x10(7) CFU of each GAS strain, the GAS472 strain, isolated from the blood of a patient suffering from streptococcal toxic shock syndrome (STSS), induced the highest severity levels and mortality rates. GAS472 led to a 100% mortality rate in CD46 Tg mice after only 168 h postinfection through the supervention of severe necrotizing fasciitis (NF) of the feet. In contrast, GAS472 led to a 10% mortality rate in non-Tg mice through the supervention of partial necrotizing cutaneous lesions of the feet. The footpad skin sections of CD46 Tg mice showed hemorrhaging and necrotic striated muscle layers in the dermis, along with the exfoliation of epidermis with intracellular edema until 48 h after s.c. infection with GAS472. Thereafter, the bacteria proliferated, reaching a 90-fold or 7-fold increase in the livers of CD46 Tg mice or non-Tg mice, respectively, for 24 h between 48 and 72 h after s.c. infection with GAS472. As a result, the infected CD46 Tg mice appeared to suffer severe liver injuries. These findings suggest that human CD46 enhanced the progression of NF in the feet and the exponential growth of bacteria in deep tissues, leading to death.

Molecular diagnosis of necrotizing fasciitis by 16S rRNA gene sequencing and superantigen gene detection.

We report the use of molecular techniques in the diagnosis of a case of culture-negative necrotizing fasciitis occurring in a 32-year-old female with no significant past medical history and who died within 36 hours of admission. Paraffin-embedded tissue sections from the popliteal fossa region obtained at autopsy showed hemorrhage, necrosis, and mild inflammation by hematoxylin and eosin staining. Tissue gram stain showed numerous gram-positive organisms arranged in clusters. The sequences of the first 500 bp of bacterial 16S rRNA gene amplified from the lesion were identical to a Lancefield group A beta-hemolytic Streptococcus pyogenes. Streptococcal pyrogenic exotoxin A and B superantigen genes were detected and an emm type 1 was determined by polymerase chain reaction and sequencing from the lesion. This confirmed the etiology of the patient's rapid deterioration with multisystem organ failure.

[Necrotising fasciitis in hyper-IgE syndrome]. / Fasciitis necrotisans hyper-IgE-szindrómában.

Hyper-IgE syndrome is a rare primary immunodeficiency disease characterized by recurrent staphylococcal skin abscesses, chronic eczematoid dermatitis, pneumonia, pneumatoceles, and extreme elevation of serum IgE. The most common pathogens are S. aureus, and C. albicans. Abnormalities of dentition, bone manifestations, and connective tissue disorders are also common features of the disease. The authors report here a 19-year-old female with hyper-IgE syndrome who developed necrotising fasciitis and toxic shock syndrome. Methicillin resistant S. aureus and S. pyogenes cultured from the skin lesions. Association of hyper-IgE syndrome with necrotising fasciitis is a rarity in the medical literature. In addition to the case report, the authors describe here the major immunologic and clinical manifestations of hyper-IgE syndrome.

An immunogenetic and molecular basis for differences in outcomes of invasive group A streptococcal infections.

The role of host genetic factors in conferring predisposition or protection in infectious diseases has become evident. Infection with group A streptococci causes a wide spectrum of disease ranging from pharyngitis to streptococcal toxic shock syndrome. The release of inflammatory cytokines triggered by streptococcal superantigens has a pivotal role in invasive streptococcal disease. However, individuals infected with the same strain can develop very different manifestations. We report here that the immunogenetics of the host influence the outcome of invasive streptococcal infection, and demonstrate the underlying mechanism for these genetic associations. Specific human leukocyte antigen class II haplotypes conferred strong protection from severe systemic disease, whereas others increased the risk of severe disease. Patients with the DRB1*1501/DQB1*0602 haplotype mounted significantly reduced responses and were less likely to develop severe systemic disease (P < 0.0001). We propose that human leukocyte antigen class II allelic variation contributes to differences in severity of invasive streptococcal infections through their ability to regulate cytokine responses triggered by streptococcal superantigens.