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.

Streptococcus pyogenes arginine and citrulline catabolism promotes infection and modulates innate immunity.

A bacterium's ability to acquire nutrients from its host during infection is an essential component of pathogenesis. For the Gram-positive pathogen Streptococcus pyogenes, catabolism of the amino acid arginine via the arginine deiminase (ADI) pathway supplements energy production and provides protection against acid stress in vitro. Its expression is enhanced in murine models of infection, suggesting an important role in vivo. To gain insight into the function of the ADI pathway in pathogenesis, the virulence of mutants defective in each of its enzymes was examined. Mutants unable to use arginine (ΔArcA) or citrulline (ΔArcB) were attenuated for carriage in a murine model of asymptomatic mucosal colonization. However, in a murine model of inflammatory infection of cutaneous tissue, the ΔArcA mutant was attenuated but the ΔArcB mutant was hyperattenuated, revealing an unexpected tissue-specific role for citrulline metabolism in pathogenesis. When mice defective for the arginine-dependent production of nitric oxide (iNOS(-/-)) were infected with the ΔArcA mutant, cutaneous virulence was rescued, demonstrating that the ability of S. pyogenes to utilize arginine was dispensable in the absence of nitric oxide-mediated innate immunity. This work demonstrates the importance of arginine and citrulline catabolism and suggests a novel mechanism of virulence by which S. pyogenes uses its metabolism to modulate innate immunity through depletion of an essential host nutrient.

Murine vaginal colonization model for investigating asymptomatic mucosal carriage of Streptococcus pyogenes.

While many virulence factors promoting Streptococcus pyogenes invasive disease have been described, specific streptococcal factors and host properties influencing asymptomatic mucosal carriage remain uncertain. To address the need for a refined model of prolonged S. pyogenes asymptomatic mucosal colonization, we have adapted a preestrogenized murine vaginal colonization model for S. pyogenes. In this model, derivatives of strains HSC5, SF370, JRS4, NZ131, and MEW123 established a reproducible, asymptomatic colonization of the vaginal mucosa over a period of typically 3 to 4 weeks' duration at a relatively high colonization efficiency. Prior treatment with estradiol prolonged streptococcal colonization and was associated with reduced inflammation in the colonized vaginal epithelium as well as a decreased leukocyte presence in vaginal fluid compared to the levels of inflammation and leukocyte presence in non-estradiol-treated control mice. The utility of our model for investigating S. pyogenes factors contributing to mucosal carriage was verified, as a mutant with a mutation in the transcriptional regulator catabolite control protein A (CcpA) demonstrated significant impairment in vaginal colonization. An assessment of in vivo transcriptional activity in the CcpA(-) strain for several known CcpA-regulated genes identified significantly elevated transcription of lactate oxidase (lctO) correlating with excessive generation of hydrogen peroxide to self-lethal levels. Deletion of lctO did not impair colonization, but deletion of lctO in a CcpA(-) strain prolonged carriage, exceeding even that of the wild-type strain. Thus, while LctO is not essential for vaginal colonization, its dysregulation is deleterious, highlighting the critical role of CcpA in promoting mucosal colonization. The vaginal colonization model should prove effective for future analyses of S. pyogenes mucosal colonization.

Catheter-associated Nocardia higoensis bacteremia in a child with acute lymphocytic leukemia.

A 23-month-old child with leukemia who was receiving chemotherapy developed fevers. Serial blood cultures from a central venous catheter and a peripheral venous site grew an organism identified by 16S rRNA gene sequencing and phenotypic analysis as Nocardia higoensis, an opportunistic organism isolated once previously from a pulmonary infection in Japan.

Severe Acute Respiratory Syndrome Coronavirus 2: Manifestations of Disease and Approaches to Treatment and Prevention in Humans.

The coronavirus disease 2019 (COVID-19) pandemic was caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This virus has challenged civilization and modern science in ways that few infectious diseases and natural disasters have previously, causing globally significant human morbidity and mortality and triggering economic downturns across financial markets that will be dealt with for generations. Despite this, the pandemic has also brought an opportunity for humanity to come together and participate in a shared scientific investigation. Clinically, SARS-CoV-2 is associated with lower mortality rates than other recently emerged coronaviruses, such as SARS-CoV and the Middle East respiratory syndrome coronavirus (MERS-CoV). However, SARS-CoV-2 exhibits efficient human-to-human spread, with transmission often occurring before symptom recognition; this feature averts containment strategies that had worked previ- ously for SARS-CoV and MERS-CoV. Severe COVID-19 disease is characterized by dysregulated inflammatory responses associated with pulmonary congestion and intravascular coagulopathy leading to pneumonia, vascular insults, and multiorgan disease. Approaches to treatment have combined supportive care with antivirals, such as remdesivir, with immunomodulatory medications, including corticosteroids and cytokine-blocking antibody therapies; these treatments have advanced rapidly through clinical trials. Innovative approaches to vaccine development have facilitated rapid advances in design, testing, and distribution. Much remains to be learned about SARS-CoV-2 and COVID-19, and further biomedical research is necessary, including comparative medicine studies in animal models. This overview of COVID-19 in humans will highlight important aspects of disease, relevant pathophysiology, underlying immunology, and therapeutics that have been developed to date.

Vertebral osteomyelitis due to Salmonella Poona in a healthy adolescent female.

We present a case of vertebral osteomyelitis in a previously healthy, adolescent Caucasian female athlete. After months of lower back pain, spinal imaging demonstrated phlegmon and suspected osteomyelitis of the L4 vertebral body. A bone biopsy was obtained, and microbiologic cultures yielded pure growth of Salmonella enterica subsp. enterica serovar Poona (S. Poona), a member of the nontyphoid Salmonella group associated with food-borne gastroenteritis in the United States. This case represents the first reported association of S. Poona with osteomyelitis and is interesting in that the infection developed in a patient without traditional risk factors for invasive Salmonella disease (i.e. sickle cell disease). This case highlights the importance of keeping a broad differential diagnosis for lower back pain and emphasizes the value of obtaining specimens for microbiologic culture to aid in diagnosing non-traditional and potentially emerging bacterial pathogens.

Recurrent Mycoplasma pneumoniae infection in a human immunodeficiency virus-positive child.

Mycoplasma pneumoniae is a frequent cause of community-acquired respiratory infections, especially in young children and adolescents. The significance of M. pneumoniae infection in HIV-positive patients, particularly children, is not well described. This report describes an HIV-positive female child with recurrent B-cell lymphoma and recurrent or relapsing pulmonary infections with M. Pneumoniae.

CcpA Coordinates Growth/Damage Balance for Streptococcus pyogenes Pathogenesis.

To achieve maximum fitness, pathogens must balance growth with tissue damage, coordinating metabolism and virulence factor expression. In the gram-positive bacterium Streptococcus pyogenes, the DNA-binding transcriptional regulator Carbon Catabolite Protein A (CcpA) is a master regulator of both carbon catabolite repression and virulence, suggesting it coordinates growth/damage balance. To examine this, two murine models were used to compare the virulence of a mutant lacking CcpA with a mutant expressing CcpA locked into its high-affinity DNA-binding conformation (CcpAT307Y). In models of acute soft tissue infection and of long-term asymptomatic mucosal colonization, both CcpA mutants displayed altered virulence, albeit with distinct growth/damage profiles. Loss of CcpA resulted in a diminished ability to grow in tissue, leading to less damage and early clearance. In contrast, constitutive DNA-binding activity uncoupled the growth/damage relationship, such that high tissue burdens and extended time of carriage were achieved, despite reduced tissue damage. These data demonstrate that growth/damage balance can be actively controlled by the pathogen and implicate CcpA as a master regulator of this relationship. This suggests a model where the topology of the S. pyogenes virulence network has evolved to couple carbon source selection with growth/damage balance, which may differentially influence pathogenesis at distinct tissues.

Contributions of CD8 T cells to the pathogenesis of mouse adenovirus type 1 respiratory infection.

CD8 T cells are key components of the immune response to viruses, but their roles in the pathogenesis of adenovirus respiratory infection have not been characterized. We used mouse adenovirus type 1 (MAV-1) to define CD8 T cell contributions to the pathogenesis of adenovirus respiratory infection. CD8 T cell deficiency in ß2m-/- mice had no effect on peak viral replication in lungs, but clearance of virus was delayed in ß2m-/- mice. Virus-induced weight loss and increases in bronchoalveolar lavage fluid total protein, IFN-γ, TNF-α, IL-10, CCL2, and CCL5 concentrations were less in ß2m-/- mice than in controls. CD8 T cell depletion had similar effects on virus clearance, weight loss, and inflammation. Deficiency of IFN-γ or perforin had no effect on viral replication or inflammation, but perforin-deficient mice were partially protected from weight loss. CD8 T cells promote MAV-1-induced pulmonary inflammation via a mechanism that is independent of direct antiviral effects.

Complete Genome Sequence of emm4 Streptococcus pyogenes MEW427, a Throat Isolate from a Child Meeting Clinical Criteria for Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus (PANDAS).

We report the complete genome assembly of the Streptococcus pyogenes type emm4 strain MEW427 (also referred to as strain UM001 in the Pediatric Acute-Onset Neuropsychiatric Syndrome [PANS] Research Consortium), a throat isolate from a child with acute-onset neuropsychiatric symptoms meeting clinical criteria for PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcus). The genome length is 1,814,455 bp with 38.51% G+C%.

Complete Genome Sequence of emm28 Type Streptococcus pyogenes MEW123, a Streptomycin-Resistant Derivative of a Clinical Throat Isolate Suitable for Investigation of Pathogenesis.

We present here the complete genome sequence of Streptococcus pyogenes type emm28 strain MEW123, a streptomycin-resistant derivative of a pediatric throat isolate. The genome length is 1,878,699 bp, with 38.29% G+C% content. The genome sequence adds value to this virulent emm28 representative strain and will aid in the investigation of streptococcal pathogenesis.

Interleukin-17A Contributes to the Control of Streptococcus pyogenes Colonization and Inflammation of the Female Genital Tract.

Postpartum women are at increased risk of developing puerperal sepsis caused by group A Streptococcus (GAS). Specific GAS serotypes, including M1 and M28, are more commonly associated with puerperal sepsis. However, the mechanisms of GAS genital tract infection are not well understood. We utilized a murine genital tract carriage model to demonstrate that M1 and M28 GAS colonization triggers TNF-α, IL-1ß, and IL-17A production in the female genital tract. GAS-induced IL-17A significantly influences streptococcal carriage and alters local inflammatory responses in two genetically distinct inbred strains of mice. An absence of IL-17A or the IL-1 receptor was associated with reduced neutrophil recruitment to the site of infection; and clearance of GAS was significantly attenuated in IL-17A(-/-) mice and Rag1(-/-) mice (that lack mature lymphocytes) but not in mice deficient for the IL-1 receptor. Together, these findings support a role for IL-17A in contributing to the control of streptococcal mucosal colonization and provide new insight into the inflammatory mediators regulating host-pathogen interactions in the female genital tract.

Is DRE essential for the follow up of prostate cancer patients? A prospective audit of 194 patients.

BACKGROUND: Prostate cancer follow up forms a substantial part of the urology outpatient workload. Nurse led prostate cancer follow up clinics are becoming more common. Routine follow-up may involve performing DRE, which may require training. OBJECTIVES: The aim of this audit was to assess the factors that influenced the change in the management of prostate cancer patients during follow up. This would allow us to pave the way towards a protocol driven follow up clinic led by nurse specialists without formal training in DRE. RESULTS: 194 prostate cancer patients were seen over a period of two months and all the patients had DRE performed on at least one occasion. The management was changed in 47 patients. The most common factor influencing this change was PSA trend. A change in DRE findings influenced advancement of the clinic visit in 2 patients. CONCLUSIONS: PSA is the most common factor influencing change in the management of these patients. Nurse specialists can run prostate cancer follow-up clinics in parallel to existing consultant clinics and reserve DRE only for those patients who have a PSA change or have onset of new symptoms. However larger studies are required involving all the subgroups of patients to identify the subgroups of patients who will require DRE routinely.

Fatal human herpesvirus 6-associated encephalitis in two boys with underlying POLG mitochondrial disorders.

BACKGROUND: Human herpesvirus 6 is a significant cause of the febrile illness roseola infantum in young children. Infection with human herpesvirus 6 typically causes a self-limited febrile illness but occasionally is associated with central nervous system manifestations, including febrile seizures and encephalitis. Host factors associated with severe manifestations of human herpesvirus 6-associated neurological disease remain poorly characterized. CASE REPORTS: We report two previously healthy young boys with human herpesvirus 6-associated encephalitis who developed a progressive, and ultimately fatal, encephalopathy with refractory movement disorder concurrent with acquisition of acute human herpesvirus 6 infection. Both children were treated with the antiviral ganciclovir without improvement of their neurological symptoms, although quantitative human herpesvirus 6 polymerase chain reaction of cerebrospinal fluid and/or blood confirmed a decline in viral load with treatment. The clinical course in both cases was most consistent with Alpers-Huttenlocher syndrome, given the intractable seizures, developmental regression, and, ultimately, death due to liver and renal failure. In support of this, postmortem analysis identified both children to be compound heterozygous for mutations in the mitochondrial polymerase γ gene, POLG. CONCLUSIONS: POLG mutations are associated with Alpers-Huttenlocher syndrome; however, no prior studies have examined the role of acute human herpesvirus 6 infection in these patients presenting with severe neurological disease. It is possible the POLG mutation phenotype was unmasked and/or exacerbated by human herpesvirus 6 infection in these two patients, potentially contributing to a more rapid clinical deterioration. This report provides new insight into a previously unrecognized association between POLG mutations and poor neurological outcome after human herpesvirus 6 infection.

Adhesin genes and serum resistance in Haemophilus influenzae type f isolates.

The incidence of invasive infections due to Haemophilus influenzae has decreased significantly in developed countries with high rates of vaccination against H. influenzae serotype b (Hib). This vaccine provides no protection against H. influenzae serotype f (Hif), typically associated with invasive infections in adults with chronic disease and/or immunodeficiency, and rarely in otherwise healthy adults and children. The specific properties of Hif associated with virulence remain largely uncharacterized. A panel of 26 Hif strains consisting of both invasive disease-associated and mucosal surface non-invasive disease-associated isolates was surveyed by DNA fingerprinting, biotyping and PCR detection of hmw1, hmw2, hsf, the hif fimbrial locus and the lipo-oligosaccharide (LOS) biosynthetic island, and assessment of ß-lactamase expression and determination of resistance to the bactericidal activity of normal adult human serum. Repetitive sequence-based PCR fingerprinting differentiated the 26 strains into three clusters, with the majority of isolates (22/26, 84.6 %) clustered into a single indistinguishable group. Most isolates (24/26, 92.3 %) were of biotype I and two isolates produced ß-lactamase with detection of a conjugative plasmid, and the isolates displayed a range of resistances to the bactericidal activity of human serum. All 26 isolates carried the adhesin hsf, 21 carried a partial hif fimbrial operon and 4 had the adhesin genes hmw1/2. A LOS biosynthetic island was detected in 20 isolates consisting of the genes lic2BC. It was concluded that Hif has many recognized virulence properties and comprises a relatively homogeneous group independent of the anatomical source from which it was isolated.

Role of lgtC in resistance of nontypeable Haemophilus influenzae strain R2866 to human serum.

We are investigating a nontypeable Haemophilus influenzae (NTHI) strain, R2866, isolated from a child with meningitis. R2866 is unusually resistant to killing by normal human serum. The serum 50% inhibitory concentration (IC50) for this strain is 18%, approaching that of encapsulated H. influenzae. R3392 is a derivative of R2866 that was found to have increased sensitivity to human serum (IC50, 1.5%). Analysis of tetrameric repeat regions within lipooligosaccharide (LOS) biosynthetic genes in both strains indicated that the glycosyltransferase gene lgtC was out of frame ("off") in most colonies of R3392 but in frame with its start codon ("on") in most colonies of the parent. We sought antigenic and biochemical evidence for modification of the LOS structure. In a whole-cell enzyme-linked immunosorbent assay, strain R3392 displayed reduced binding of the Galalpha1,4Gal-specific monoclonal antibody 4C4. Mass spectrometry analysis of LOS from strain R2866 indicated that the primary oligosaccharide glycoform contained four heptose and four hexose residues, while that of R3392 contained four heptose and three hexose residues. We conclude that the R2866 lgtC gene encodes a galactosyltransferase involved in synthesis of the 4C4 epitope, as in other strains, and that expression of lgtC is associated with the high-level serum resistance that has been observed for this strain. This is the first description of the genetic basis of high-level serum resistance in NTHI, as well as the first description of LOS composition in an NTHI strain for which the complete genome sequence has been determined.

Inactivation of deoxyadenosine methyltransferase (dam) attenuates Haemophilus influenzae virulence.

Mutants in deoxyadenosine methyltransferase (dam) from many Gram-negative pathogens suggest multiple roles for Dam methylase: directing post-replicative DNA mismatch repair to the correct strand, guiding the temporal control of DNA replication and regulating the expression of multiple genes (including virulence factors) by differential promoter methylation. Dam methylase (HI0209) in strain Rd KW20 was inactivated in Haemophilus influenzae strains Rd KW20, Strain 12 and INT-1; restriction with Dam methylation-sensitive enzymes DpnI and DpnII confirmed the absence of Dam methylation, which was restored by complementation with a single copy of dam ectopically expressed in cis. Despite the lack of increased mutation frequency, the dam mutants had a 2-aminopurine-susceptible phenotype that could be suppressed by secondary mutations in mutS, suggesting a role for Dam in H. influenzae DNA mismatch repair. Invasion of human brain microvascular endothelial cells (HBMECs) and human respiratory epithelial cells (NCI-H292) by the dam mutants was significantly attenuated in all strains, suggesting the absence of a Dam-regulated event necessary for uptake or invasion of host cells. Intracellular replication was inhibited only in the Strain 12 dam mutant, whereas in the infant rat model of infection, the INT-1 dam mutant was less virulent. Dam activity appears to be necessary for both in vitro and in vivo virulence in a strain-dependent fashion and may function as a regulator of gene expression including virulence factors.

Hypermutable Haemophilus influenzae with mutations in mutS are found in cystic fibrosis sputum.

Hypermutable bacterial pathogens exist at surprisingly high prevalence and benefit bacterial populations by promoting adaptation to selective environments, including resistance to antibiotics. Five hundred Haemophilus influenzae isolates were screened for an increased frequency of mutation to resistance to rifampicin, nalidixic acid and spectinomycin: of the 14 hypermutable isolates identified, 12 were isolated from cystic fibrosis (CF) sputum. Analysis by enterobacterial repetitive intergenic consensus (ERIC)-PCR and ribotyping identified eight distinct genetic fingerprints. The hypermutable phenotype of seven of the eight unique isolates was associated with polymorphisms in conserved sites of mutS. Four of the mutant mutS alleles were cloned and failed to complement the mutator phenotype of a mutS : : TSTE mutant of H. influenzae strain Rd KW20. Antibiotic susceptibility testing of the hypermutators identified one beta-lactamase-negative ampicillin-resistant (BLNAR) isolate with two isolates producing beta-lactamase. Six isolates from the same patient with CF, with the same genetic fingerprint, were clonal by multilocus sequence typing (MLST). In this clone, there was an evolution to higher MIC values for the antibiotics administered to the patient during the period in which the strains were isolated. Hypermutable H. influenzae with mutations in mutS are prevalent, particularly in the CF lung environment, and may be selected for and maintained by antibiotic pressure.