Whole genome sequencing of Mycobacterium tuberculosis reveals slow growth and low mutation rates during latent infections in humans

Very little is known about the growth and mutation rates of Mycobacterium tuberculosis during latent infection in humans. However, studies in rhesus macaques have suggested that latent infections have mutation rates that are higher than that observed during active tuberculosis disease. Elevated mutation rates are presumed risk factors for the development of drug resistance. Therefore, the investigation of mutation rates during human latency is of high importance. We performed whole genome mutation analysis of M. tuberculosis isolates from a multi-decade tuberculosis outbreak of the New Zealand Rangipo strain. We used epidemiological and phylogenetic analysis to identify four cases of tuberculosis acquired from the same index case. Two of the tuberculosis cases occurred within two years of exposure and were classified as recently transmitted tuberculosis. Two other cases occurred more than 20 years after exposure and were classified as reactivation of latent M. tuberculosis infections. Mutation rates were compared between the two recently transmitted pairs versus the two latent pairs. Mean mutation rates assuming 20 hour generation times were 5.5X10⁻¹⁰ mutations/bp/generation for recently transmitted tuberculosis and 7.3X10⁻¹¹ mutations/bp/generation for latent tuberculosis. Generation time versus mutation rate curves were also significantly higher for recently transmitted tuberculosis across all replication rates (p = 0.006). Assuming identical replication and mutation rates among all isolates in the final two years before disease reactivation, the u20hr mutation rate attributable to the remaining latent period was 1.6×10⁻¹¹ mutations/bp/generation, or approximately 30 fold less than that calculated during the two years immediately before disease. Mutations attributable to oxidative stress as might be caused by bacterial exposure to the host immune system were not increased in latent infections. In conclusion, we did not find any evidence to suggest elevated mutation rates during tuberculosis latency in humans, unlike the situation in rhesus macaques

PCC oral history tape

Oral history interview with Kathy Stranlund and Coley Meradier about their relationship with Pacific Bible Seminary (Pacific Christian College) in the 1930s and about Dr. Hirst (President of Pacific Bible Seminary)

Peptide Mimotopes Selected from a Random Peptide Library for Diagnosis of Epstein-Barr Virus Infection

Epstein-Barr virus (EBV) is a ubiquitous, worldwide infectious agent that causes infectious mononucleosis, affecting >90% of the world's population. Currently, enzyme-linked immunosorbent assay, mostly with purified preparations of EBV cell extracts to capture immunoglobulin M (IgM) antibodies in patients' serum, is used for primary diagnosis. Our objective was to determine whether a small set of peptides could contain sufficient immunogenic information to replace solid-phase antigens in EBV diagnostics. Using monoclonal antibodies, we selected four peptides that mimic different epitopes of EBV from a phage-displayed random peptide library. To assess their diagnostic value, we screened a panel of 62 individual EBV IgM sera for their reactivities with the peptides alone. For all peptides, there was a clear distinction between the EBV-positive and the EBV-negative samples, resulting in 100% specificity. The sensitivities were 88%, 85%, 71%, and 54% for peptides F1, A3, gp125, and A2, respectively. Any combination of peptides increased the sensitivity, indicating that individual peptides react with different subsets of antibodies. Furthermore, when the F1 and the gp125 peptides were coupled to bovine serum albumin and screened against 216 serum samples, there were dramatic improvements in sensitivities (95% and 92%, respectively) and little cross-reactivity with the other peptides encountered during acute viral infections, including rheumatoid factor. This study shows the potential for the use of peptide mimotopes as alternatives to the complex antigens used in current serodiagnostics for EBV infection

Mimotopes of Apical Membrane Antigen 1: Structures of Phage-Derived Peptides Recognized by the Inhibitory Monoclonal Antibody 4G2dc1 and Design of a More Active Analogue

Apical membrane antigen 1 (AMA1) of the malaria parasite Plasmodium falciparum is an integral membrane protein that plays a key role in merozoite invasion of host erythrocytes. A monoclonal antibody, 4G2dc1, recognizes correctly folded AMA1 and blocks merozoite invasion. Phage display was used to identify peptides that bind to 4G2dc1 and mimic an important epitope of AMA1. Three of the highest-affinity binders—J1, J3, and J7—were chosen for antigenicity and immunogenicity studies. J1 and J7 were found to be true antigen mimics since both peptides generated inhibitory antibodies in rabbits (J. L. Casey et al., Infect. Immun. 72:1126-1134, 2004). In the present study, the solution structures of all three mimotopes were investigated by nuclear magnetic resonance spectroscopy. J1 adopted a well-defined region of structure, which can be attributed in part to the interactions of Trp11 with surrounding residues. In contrast, J3 and J7 did not adopt an ordered conformation over the majority of residues, although they share a region of local structure across their consensus sequence. Since J1 was the most structured of the peptides, it provided a template for the design of a constrained analogue, J1cc, which shares a structure similar to that of J1 and has a disulfide-stabilized conformation around the Trp11 region. J1cc binds with greater affinity to 4G2dc1 than does J1. These peptide structures provide the foundation for a better understanding of the complex conformational nature of inhibitory epitopes on AMA1. With its greater conformational stability and higher affinity for AMA1, J1cc may be a better in vitro correlate of immunity than the peptides identified by phage display

Number of single nucleotide polymorphisms differences between each strain.

<p>Number of single nucleotide polymorphisms differences between each strain.</p

Epidemiological relationships among 11 New Zealand <i>M. tuberculosis</i> cases.

<p>Chronological representation of different subjects in a New Zealand TB outbreak. Each square represent a subject at the time of the TB diagnosis. Broken lines represent known close direct contact with the initial index case “X” during X's period of infectiousness. Solid lines show assumed connections between a case of presumed reactivation (C1 to C2) and a case of potential child-parent transmission (A to T).</p

Evolutionary history of <i>M. tuberculosis</i> strains isolated from a cluster of cases in New Zealand.

<p>Relationships were inferred by examining parsimony and nonparsimony informative single nucleotide polymorphisms (SNPs). <b>Panel A</b>: parallelogram of network analysis using 34 SNPs including five homoplastic SNPs. <b>Panel B</b>: Evolutionary tree using the same 34 SNPs as in A rooted on <i>M. tuberculosis</i> H₃₇Rv analyzed using Neighbor-joining method. Panel C. Evolutionary tree examining 29 nonparsimony SNPs using the Neighbor-joining method. The evolutionary distances represent the number of SNP differences per strain.</p

Single nucleotide polymorphisms identified in sequenced isolates.

1<p>reference genome.</p

In vivo mutation rates in <i>M. tuberculosis</i> strains for generation times ranging from 18 to 240 hours.

<p>The mutation rate for each of the <i>M. tuberculosis</i> isolates was estimated using equation (1) in Ford <i>et al</i>, and calculated as untis of mutation/bp/generation <b>Panel A</b>: The mutation rate for each <i>M. tuberculosis</i> isolate in the study. Isolate C1 and E are from recent infection while strains O and S from reactivation after a prolonged period of latency. The yellow areas represent 95% confidence intervals. <b>Panel B</b>: Isolates with recent infection (E and C1) and reactivation after prolonged latency (O and S) were combined to obtain overall estimates of mutation rates of recent and latent-reactivated disease. The yellow areas represent 95% confidence intervals.</p