Biochemical and functional characterization of the tapasin/ERp57 conjugate

Recognition of MHC class I/peptide complexes is required for the generation of CD8+ T cell responses. Peptide loading onto MHC class I/beta2m dimers occurs in the ER and involves both specific proteins and cellular chaperones. Tapasin is essential for peptide loading onto most MHC class I alleles, and it forms a mixed disulfide with the glycoprotein specific oxidoreductase ERp57. I have characterized the biochemical requirements for tapasin/ERp57 conjugate formation and addressed potential functions for ERp57 in peptide loading. Tapasin specifically recruits ERp57 into a mixed disulfide at the expense of free ERp57 in the ER. Other components of the MHC class I peptide loading complex are not required for conjugate formation, and, in contrast to models of glycoprotein folding, conjugate formation does not require the generation of monoglucosylated glycans.Once associated, tapasin has evolved to inhibit the reductase activity of the ERp57 a domain leading to the retention of ERp57 in the loading complex over the course of normal peptide loading. In contrast, calreticulin undergoes cycles of binding and release, and its presence in the loading complex is dependent upon MHC class I. ERp57 is a core structural component of the MHC class I loading complex, and it is permanently sequestered there by tapasin.Finally, I have further characterized cells expressing a tapasin mutant unable to form the conjugate. Loading complex assembly is impaired in these cells, and peptide loading is inefficiently catalyzed by this mutant. Additionally, ERp57 binding stabilizes the structural integrity of tapasin. When endogenous ERp57 expression was suppressed using RNAi, all residual ERp57 was bound to tapasin, and both protein disulfide isomerase and ERp72, other ER resident oxidoreductases, formed mixed disulfides with tapasin. When redox mutant ERp57 proteins were over-expressed in these cells, only slight changes were seen in MHC class I trafficking. My data indicate that ERp57 is a key structural component of the MHC class I loading complex and likely does not exhibit any redox activity in this context. The key features of ERp57 responsible for its function remain unknown

Comparison of Disk Diffusion and Etest Methods to Determine the Susceptibility of Staphylococcus aureus

Fusidic acid is a common therapy for staphylococcal infections in Saudi Arabia, but reports have suggested high rates of resistance among clinical isolates. Susceptibility testing of S. aureus to fusidic acid is further complicated by the lack of consensus on mean inhibitory concentrations (MIC) and disk diffusion cutoffs to determine resistance. The purpose of this study was to determine the correlation between disk diffusion and Etest determined MIC susceptibility results in clinical isolates of S. aureus from a large academic hospital in Riyadh, Saudi Arabia. Our data demonstrate excellent correlation between Etest determined MIC and disk diffusion susceptibility data, using either previously proposed zone sizes of ≥21 mm as susceptible and ≤18 mm as resistant or the EUCAST recommended zone size of ≤24 mm for resistance, in an area with relatively high rates of fusidic acid resistance

Accelerated SARS-CoV-2 Intrahost Evolution Leading to Distinct Genotypes During Chronic Infection

The chronic infection hypothesis for novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant emergence is increasingly gaining credence following the appearance of Omicron. Here, we investigate intrahost evolution and genetic diversity of lineage B.1.517 during a SARS-CoV-2 chronic infection lasting for 471 days (and still ongoing) with consistently recovered infectious virus and high viral genome copies. During the infection, we find an accelerated virus evolutionary rate translating to 35 nucleotide substitutions per year, approximately 2-fold higher than the global SARS-CoV-2 evolutionary rate. This intrahost evolution results in the emergence and persistence of at least three genetically distinct genotypes, suggesting the establishment of spatially structured viral populations continually reseeding different genotypes into the nasopharynx. Finally, we track the temporal dynamics of genetic diversity to identify advantageous mutations and highlight hallmark changes for chronic infection. Our findings demonstrate that untreated chronic infections accelerate SARS-CoV-2 evolution, providing an opportunity for the emergence of genetically divergent variants

Comparative Transmissibility of SARS-CoV-2 Variants Delta and Alpha in New England, USA

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant quickly rose to dominance in mid-2021, displacing other variants, including Alpha. Studies using data from the United Kingdom and India estimated that Delta was 40-80% more transmissible than Alpha, allowing Delta to become the globally dominant variant. However, it was unclear if the ostensible difference in relative transmissibility was due mostly to innate properties of Delta\u27s infectiousness or differences in the study populations. To investigate, we formed a partnership with SARS-CoV-2 genomic surveillance programs from all six New England US states. By comparing logistic growth rates, we found that Delta emerged 37-163% faster than Alpha in early 2021 (37% Massachusetts, 75% New Hampshire, 95% Maine, 98% Rhode Island, 151% Connecticut, and 163% Vermont). We next computed variant-specific effective reproductive numbers and estimated that Delta was 58-120% more transmissible than Alpha across New England (58% New Hampshire, 68% Massachusetts, 76% Connecticut, 85% Rhode Island, 98% Maine, and 120% Vermont). Finally, using RT-PCR data, we estimated that Delta infections generate on average ∼6 times more viral RNA copies per mL than Alpha infections. Overall, our evidence indicates that Delta\u27s enhanced transmissibility could be attributed to its innate ability to increase infectiousness, but its epidemiological dynamics may vary depending on the underlying immunity and behavior of distinct populations

Combining genomic and epidemiological data to compare the transmissibility of SARS-CoV-2 variants Alpha and Iota.

SARS-CoV-2 variants shaped the second year of the COVID-19 pandemic and the discourse around effective control measures. Evaluating the threat posed by a new variant is essential for adapting response efforts when community transmission is detected. In this study, we compare the dynamics of two variants, Alpha and Iota, by integrating genomic surveillance data to estimate the effective reproduction number (Rt) of the variants. We use Connecticut, United States, in which Alpha and Iota co-circulated in 2021. We find that the Rt of these variants were up to 50% larger than that of other variants. We then use phylogeography to show that while both variants were introduced into Connecticut at comparable frequencies, clades that resulted from introductions of Alpha were larger than those resulting from Iota introductions. By monitoring the dynamics of individual variants throughout our study period, we demonstrate the importance of routine surveillance in the response to COVID-19

Comparative transmissibility of SARS-CoV-2 variants Delta and Alpha in New England, USA.

The SARS-CoV-2 Delta variant rose to dominance in mid-2021, likely propelled by an estimated 40%-80% increased transmissibility over Alpha. To investigate if this ostensible difference in transmissibility is uniform across populations, we partner with public health programs from all six states in New England in the United States. We compare logistic growth rates during each variant\u27s respective emergence period, finding that Delta emerged 1.37-2.63 times faster than Alpha (range across states). We compute variant-specific effective reproductive numbers, estimating that Delta is 63%-167% more transmissible than Alpha (range across states). Finally, we estimate that Delta infections generate on average 6.2 (95% CI 3.1-10.9) times more viral RNA copies per milliliter than Alpha infections during their respective emergence. Overall, our evidence suggests that Delta\u27s enhanced transmissibility can be attributed to its innate ability to increase infectiousness, but its epidemiological dynamics may vary depending on underlying population attributes and sequencing data availability

Functional Relationship between Protein Disulfide Isomerase Family Members during the Oxidative Folding of Human Secretory Proteins

We systematically depleted PDI family members and show that whereas ERp72 and P5 contributed minimally to oxidative protein folding, PDI and ERp57 were the predominant catalysts. Depletion of PDI or ERp57 alone modestly delayed folding, but depletion of both led to generalized protein misfolding and degradation

Clinical implications of Paracoccus yeeii bacteremia in a patient with decompensated cirrhosis

Infections in patients with cirrhosis are common among those who develop variceal hemorrhage. Prophylactic antimicrobial treatment with third generation cephalosporins is recommended in patients with advanced cirrhosis and gastrointestinal hemorrhage. However no infectious source is identified in up to 50% of patients with cirrhosis and clinical sepsis. We report the first case of Paracoccus yeeii bacteremia in a patient with decompensated cirrhosis who presented with variceal hemorrhage. This rare gram negative organism that occurs naturally in the soil has been difficult to isolate until recent technological advances and may not be susceptible to third generation cephalosporins. Our case reinforces the challenges in isolating rare infections in patients with cirrhosis, the need to consider uncommon organisms in infected but culture negative patients with cirrhosis, and the importance of optimizing antimicrobials to reduce the incidence of drug resistant organisms