bBacillus anthracis, the causative agent of anthrax, replicates as chains of vegetative cells by regulating the separation of septal peptidoglycan. Surface (S)-layer proteins and associated proteins (BSLs) function as chain length determinants and bind to the secondary cell wall polysaccharide (SCWP). In this study, we identified the B. anthracis lcpD mutant, which displays increased chain length and S-layer assembly defects due to diminished SCWP attachment to peptidoglycan. In contrast, the B. anthracis lcpB3 variant displayed reduced cell size and chain length, which could be attributed to increased deposition of BSLs. In other bacteria, LytR-CpsA-Psr (LCP) proteins attach wall teichoic acid (WTA) and polysaccharide capsule to peptidoglycan. B. anthracis does not synthesize these polymers, yet its genome encodes six LCP homologues, which, when expressed in S. aureus, promote WTA attachment. We propose a model whereby B. anthracis LCPs promote attachment of SCWP precursors to discrete locations in the peptidoglycan, enabling BSL assembly and regulated separation of septal peptidoglycan. Bacillus anthracis, the causative agent of anthrax, is a sporeforming, Gram-positive bacterium that germinates in host infected tissues and replicates as elongated chains of vegetative forms (1, 2). This unique growth pattern appears to be caused by the regulated separation of septal peptidoglycan, which generates bacterial chains whose mere size precludes clearance by host phagocytes (3-5). The genetic determinants for B. anthracis chain formation are conserved among pathogenic species of the Bacillus cereus group and, as demonstrated with B. cereus G9241, likely contribute to disease pathogenesis (6, 7). Hallmarks of pathogenic Bacillus species are virulence plasmids, pXO-1 and pXO-2 in B. anthracis (8, 9), providing for toxin production and capsulation (10, 11) as well as the chromosomally encoded surface (S)-layer locus (12). Two S-layer proteins of B. anthracis, Sap and EA1, are endowed with S-layer homology (SLH) domains, which retain these proteins in the bacterial envelope by binding to the secondary cell wall polysaccharide (SCWP) (13-15). S-layer protein crystallization domains, responsible for the spontaneous assembly of these polypeptides into a paracrystalline array (16), form a twodimensional lattice that can be thought of as bacterial integument (17)(18)(19).The structural genes of S-layer proteins, sap and eag, are flanked by genes encoding determinants for S-layer protein secretion (secA2 and slaP) (12) or pyruvylation (csaB) (14) as well as acetylation of the SCWP (patA1/2 and patB1/2) (20). CsaB-mediated pyruvylation of the terminal N-acetylmannosamine (ManNAc) of the SCWP (21), with the repeat struc- (22), is a prerequisite for the assembly of S-layer proteins and 22 B. anthracis S-layer-associated proteins (BSLs) (14). PatAB1/2-mediated acetylation of SCWP molecules affects the assembly of EA1 and of some but not all BSLs (20). Recent studies have begun to identify genes for SCWP synthesis, which, unlike py...

Objectives Serologic detection of prior severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is needed for definition of convalescent plasma donors, for confounding SARS-CoV-2 presentation, and for seroprevalence studies. Reliable serologic assays with independent validation are required. Methods Six SARS-CoV-2 antibody assays from Beckman Coulter, Euroimmun (IgG, IgA), Roche, and Siemens (Centaur, Vista) were assessed for specificity (n = 184), sensitivity (n = 154), and seroconversion in a defined cohort with clinical correlates and molecular SARS-CoV-2 results. Results Assay specificity was 99% or greater for all assays except the Euroimmun IgA (95%). Sensitivity at more than 21 days from symptom onset was 84%, 95%, 72%, 98%, 67%, and 96% for Beckman Coulter, Centaur, Vista, Roche, Euroimmun IgA, and Euroimmun IgG, respectively. Average day of seroconversion was similar between assays (8-10 d), with 2 patients not producing nucleocapsid antibodies during hospitalization. Conclusions SARS-CoV-2 nucleocapsid antibodies may be less reliably produced early in disease than spike protein antibodies. Assessment of convalescent plasma donors at more than 30 days from symptom onset and seroprevalence studies should use assays with defined sensitivity at time points of interest because not all assays detected antibodies reliably at more than 30 days.

Background The burden of COVID-19 is poorly understood in pediatric patients due to frequent asymptomatic and mild presentations. Additionally, the disease prevalence in pediatric immunocompromised patients remains unknown. Methods This cross-sectional study tested convenience samples from pediatric patients who had clinically indicated lab work collected and an immunocompromising condition, including oncologic diagnoses, solid organ transplant, bone marrow transplant, primary immunodeficiency, and rheumatologic conditions or inflammatory bowel disease on systemic immunosuppression, for the presence of antibodies to SARS-CoV-2. Results We tested sera from 485 children and observed SARS-CoV-2 seroprevalence of 1.0% (CI 95%: 0.3-2.4%). Two patients were positive by NP swab RT-PCR, but only one seroconverted. Patients with oncologic diagnoses or solid organ transplant were most likely to be tested for COVID-19 when presenting with respiratory illness as compared to other groups. Conclusions Seroprevalence of antibodies to SARS-CoV-2 in immunocompromised children was similar to that of an immunocompetent pediatric population (0.6%, CI 95%: 0.3-1.1%), suggesting an adequate antibody response. However, none of the patients who tested positive for antibodies or via NP RT-PCR had more than a mild illness course and two patients did not have any reported illness, suggesting that SARS-CoV-2 may not cause a worse clinical outcome in immunosuppressed children, in contrast to immunocompromised adults.

Bacillus anthracis elaborates a linear secondary cell wall polysaccharide (SCWP) that retains surface (S)-layer and associated proteins via their S-layer homology (SLH) domains. The SCWP is comprised of trisaccharide repeats [¡4)-␤-ManNAc-(1¡4)-␤-GlcNAc-(1¡6)-␣-GlcNAc-(1¡]and tethered via acid-labile phosphodiester bonds to peptidoglycan. Earlier work identified UDP-GlcNAc 2-epimerases GneY (BAS5048) and GneZ (BAS5117), which act as catalysts of ManNAc synthesis, as well as a polysaccharide deacetylase (BAS5051), as factors contributing to SCWP synthesis. Here, we show that tagO (BAS5050), which encodes a UDP-N-acetylglucosamine:undecaprenyl-P N-acetylglucosaminyl 1-P transferase, the enzyme that initiates the synthesis of murein linkage units, is required for B. anthracis SCWP synthesis and S-layer assembly. Similar to gneY-gneZ mutants, B. anthracis strains lacking tagO cannot maintain cell shape or support vegetative growth. In contrast, mutations in BAS5051 do not affect B. anthracis cell shape, vegetative growth, SCWP synthesis, or S-layer assembly. These data suggest that TagO-mediated murein linkage unit assembly supports SCWP synthesis and attachment to the peptidoglycan via acid-labile phosphodiester bonds. Further, B. anthracis variants unable to synthesize SCWP trisaccharide repeats cannot sustain cell shape and vegetative growth. IMPORTANCEBacillus anthracis elaborates an SCWP to support vegetative growth and envelope assembly. Here, we show that some, but not all, SCWP synthesis is dependent on tagO-derived murein linkage units and subsequent attachment of SCWP to peptidoglycan. The data implicate secondary polymer modifications of peptidoglycan and subcellular distributions as a key feature of the cell cycle in Gram-positive bacteria and establish foundations for work on the molecular functions of the SCWP and on inhibitors with antibiotic attributes.T he cell wall envelope of Bacillus anthracis, the causative agent of anthrax, is comprised of peptidoglycan and its attached secondary cell wall polysaccharide (SCWP) (1). The SCWP retains two surface (S)-layer proteins, surface array protein (Sap) and extractable antigen 1 (EA1) (2), as well as 22 S-layer-associated proteins whose S-layer homology (SLH) domains associate with ketal-pyruvylated SCWP (3, 4). Unlike nonpathogenic Bacillus spp., for example, Bacillus subtilis and Bacillus cereus AHU1030, B. anthracis does not synthesize wall teichoic acid (WTA) (5, 6). Nevertheless, B. anthracis expresses functional tagO and tagA genes, whose products provide for the synthesis of murein linkage units [P-GlcNAc-(4¡1)-␤-ManNAc-R] that are phosphodiester linked to the C 6 -hydroxyl of N-acetylmuramic acid in the repeating disaccharide of peptidoglycan [MurNAc(P-GlcNAc-ManNAc-R)-GlcNAc] (4). In B. subtilis, -R represents wall teichoic acid, i.e., either polyglycerol-phosphate or polyribitol-phosphate (7). Here, we test the hypothesis that in B. anthracis the SCWP is attached via the murine linkage unit and that the tagO gene is required for vegetati...

Bacillus anthracis, a spore-forming pathogen, replicates as chains of vegetative cells by regulating the separation of septal peptidoglycan. Surface (S)-layer proteins and B. anthracis S-layer-associated proteins (BSLs) function as chain length determinants and are assembled in the envelope by binding to the secondary cell wall polysaccharide (SCWP). B. anthracis expresses six different genes encoding LytR-CpsA-Psr (LCP) enzymes (lcpB1 to -4, lcpC, and lcpD), which when expressed in Staphylococcus aureus promote attachment of wall teichoic acid to peptidoglycan. Mutations in B. anthracis lcpB3 and lcpD cause aberrations in cell size and chain length that can be explained as discrete defects in SCWP assembly; however, the function of the other lcp genes is not known. By deleting combinations of lcp genes from the B. anthracis genome, we generated variants with single lcp genes. B. anthracis expressing lcpB3 alone displayed physiological cell size, vegetative growth, spore formation, and S-layer assembly. Strains expressing lcpB1 or lcpB4 displayed defects in cell size and shape, S-layer assembly, and spore formation yet sustained vegetative growth. In contrast, the lcpB2 strain was unable to grow unless the gene was expressed from a multicopy plasmid (lcpB2 ؉؉ ), and variants expressing lcpC or lcpD displayed severe defects in growth and cell shape. The lcpB2 ؉؉ , lcpC, or lcpD strains supported neither S-layer assembly nor spore formation. We propose a model whereby LCP enzymes fulfill partially overlapping functions in transferring SCWP molecules to discrete sites within the bacterial envelope. IMPORTANCEProducts of genes essential for bacterial envelope assembly represent targets for antibiotic development. The LytR-CpsA-Psr (LCP) enzymes tether bactoprenol-linked intermediates of secondary cell wall polymers to the C6 hydroxyl of N-acetylmuramic acid in peptidoglycan; however, the role of LCPs as a target for antibiotic therapy is not defined. We show here that LCP enzymes are essential for the cell cycle, vegetative growth, and spore formation of Bacillus anthracis, the causative agent of anthrax disease. Furthermore, we assign functions for each of the six LCP enzymes, including cell size and shape, vegetative growth and sporulation, and S-layer and S-layer-associated protein assembly. Bacillus anthracis, a Gram-positive spore-forming bacterium, infects and replicates within vertebrates, thereby precipitating anthrax disease. Without therapy, anthrax can be fatal, which triggers formation of infectious B. anthracis spores within the carcass (1, 2). During vegetative replication, B. anthracis grows as chains of rod-shaped bacteria that are tethered at septal peptidoglycan, a developmental program that protects bacteria from engulfment by phagocytes (3, 4). Earlier work identified the S-layer protein Sap, the S-layer-associated protein BslO, and the CsaB pyruvyltransferase as determinants of B. anthracis chain length (3, 5, 6). Both S-layer proteins (Sap and EA1) and BslO are endowed with three S-laye...

Background While it is presumed that immunosuppressed patients, such as solid organ transplant recipients on immunosuppression, are at greater risk from SARS-CoV-2 infection than the general population, the antibody response to infection in this patient population has not been studied. Methods In this report, we follow the anti-SARS-CoV-2 antibody levels in patients with COVID-19 who are undergoing exogenous immunosuppression. Specifically, we studied the antibody response of 3 solid organ transplant recipient patients, 3 patients who take daily inhaled fluticasone, and a patient on etanercept and daily inhaled fluticasone, and compared them to 5 patients not on exogenous immunosuppression. Results We found that the solid organ transplant patients on full immunosuppression are at risk of having a delayed antibody response and poor outcome. We did not find evidence that inhaled steroids or etanercept predispose patients to delayed immune response to SARS-CoV-2.  Conclusion The data presented here suggest that solid organ transplant recipients may be good candidates for early targeted intervention against SARS-CoV-2.

Aims: Ossifying fibromyxoid tumor (OFMT) is a rare enigmatic tumor of uncertain differentiation that can be classified as typical, atypical, and malignant subtypes based on cellularity, nuclear grade, and mitotic activity. The majority of OFMTs, regardless of the risk of malignancy, harbor genetic translocations. We report two malignant OFMTs, including one with evidence of dedifferentiation, with novel genefusions. Methods and Results: Case 1 was a 63-year-old male with a dedifferentiated OFMT arising in the right wrist, while case 2 was a 41-year-old male with a malignant OFMT presenting as a posterior mediastinal mass. Case 2 showed multifocal expression with EMA and synaptophysin, while desmin and S100 were absent in both tumors. NGS sequencing studies detected PHF1:: FOXR1 and PHF1::FOXR2 gene fusions in cases 1 and 2, respectively. Despite aggressive regimens, both progressed with wide spread metastases resulting in death within six years of diagnosis. Conclusions: We expand the genetic spectrum of OFMTs with two novel gene fusions, PHF1::FOXR1 and PHF1::FOXR2. These cases confirm the previously reported tendencies for OFMTs with rare variant fusions to demonstrate malignant behavior, unusual morphology, and non-specific immunophenotype.

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