Cryptococcus neoformans is a major life-threatening fungal pathogen. In response to the stress of the host environment, C. neoformans produces large polyploid titan cells. Titan cell production enhances the virulence of C. neoformans, yet whether the polyploid aspect of titan cells is specifically influential remains unknown. We show that titan cells were more likely to survive and produce offspring under multiple stress conditions than typical cells and that even their normally sized daughters maintained an advantage over typical cells in continued exposure to stress. Although polyploid titan cells generated haploid daughter cell progeny upon in vitro replication under nutrient-replete conditions, titan cells treated with the antifungal drug fluconazole produced fluconazole-resistant diploid and aneuploid daughter cells. Interestingly, a single titan mother cell was capable of generating multiple types of aneuploid daughter cells. The increased survival and genomic diversity of titan cell progeny promote rapid adaptation to new or high-stress conditions.

Accumulating evidence has shown the protective role of CD8+ T cells in vaccine-induced immunity against Mycobacterium tuberculosis (Mtb) despite controversy over their role in natural immunity. However, the current vaccine BCG is unable to induce sufficient CD8+ T cell responses, especially in the lung. Sendai virus, a respiratory RNA virus, is here engineered firstly as a novel recombinant anti-TB vaccine (SeV85AB) that encodes Mtb immuno-dominant antigens, Ag85A and Ag85B. A single mucosal vaccination elicited potent antigen-specific T cell responses and a degree of protection against Mtb challenge similar to the effect of BCG in mice. Depletion of CD8+ T cells abrogated the protective immunity afforded by SeV85AB vaccination. Interestingly, only SeV85AB vaccination induced high levels of lung-resident memory CD8+ T (TRM) cells, and this led to a rapid and strong recall of antigen-specific CD8+ T cell responses against Mtb challenge infection. Furthermore, when used in a BCG prime-SeV85AB boost strategy, SeV85AB vaccine significantly enhanced protection above that seen after BCG vaccination alone. Our findings suggest that CD8+ TRM cells that arise in lungs responding to this mucosal vaccination might help to protect against TB, and SeV85AB holds notable promise to improve BCG’s protective efficacy in a prime-boost immunization regimen.

Rapid detection of drug resistance in Mycobacterium tuberculosis is essential for efficient treatment and control of this pathogen. The amplification refractory mutation system (ARMS) was used to detect mutations in the rifampin resistance-determining region of the rpoB gene. A total of 39 rifampin-resistant M. tuberculosis isolates in Shanghai were analyzed by this assay, resulting in 92.3% sensitivity (36 of 39) and 87.2% concordance (34 of 39) relative to DNA sequencing, by which 41 mutations of 11 different types, including 9 point mutations and 2 deletions, were identified in the rpoB gene. The most frequent mutations were those associated with codon 531 (21 of 39 [53.8%]) and codon 526 (9 of 39 [23.1%]). The results suggest that the ARMS assay is rapid and simple to implement and could be performed for detection of rifampin resistance in M. tuberculosis to complement conventional culture-based methods.Tuberculosis (TB), though curable, still remains a major public health concern worldwide. According to the report of the World Health Organization, about one-third of the world's population (1.86 billion people) are infected with Mycobacterium tuberculosis and are at risk of having the infection develop into clinical TB. Approximately 8 million new cases occur each year, resulting in 3 million deaths around the world (15,19). It has been estimated that among 6 million active TB patients at present, the disease causes 250,000 deaths every year in China (28). Furthermore, control of TB has been further complicated by the emergence of multidrug-resistant (MDR) M. tuberculosis strains and the human immunodeficiency virus epidemic (4, 9).Rifampin, introduced in 1971, has proven to be one of the most potent antituberculosis agents (2). Rifampin is an effective bactericidal against M. tuberculosis, interacting with DNAdependent RNA polymerase to inhibit transcription and elongation of RNA (11,12), and the use of this drug has greatly shortened the duration of chemotherapy. The molecular mechanism of rifampin resistance in M. tuberculosis was first characterized in 1993 (21). Ninety-six percent of rifampin-resistant (Rif r ) M. tuberculosis strains possess genetic alterations within an 81-bp rifampin resistance-determining region (RRDR) in the rpoB gene (16, 18), corresponding to codons 507 to533 (Escherichia coli numbering system). In addition, rifampin resistance can be assumed to be a surrogate marker for MDR TB, since more than 90% of Rif r isolates are also isoniazid resistant (6).Early diagnosis of TB and rapid detection of rifampin resistance are essential for efficient treatment and control of M. tuberculosis. However, culture-based methods for detection of M. tuberculosis infection and testing of drug susceptibility usually take more than 1 month. Although several different genotypic methods, such as PCR single-strand conformational length polymorphism (21, 22), dideoxy fingerprinting (7), heteroduplex analysis (26), and DNA sequencing (13), have been used for analysis of rpoB gene muations associated with r...

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The situation of tuberculosis (TB) is very severe in China. New therapeutic agents or regimens to treat TB are urgently needed. In this study, Mycobacterium tuberculosis-infected mice were given immunotherapy intramuscularly with Ag85A/B chimeric DNA or saline, plasmid vector pVAX1, or Mycobacterium vaccae vaccine. The mice treated with Ag85A/B chimeric DNA showed significantly higher numbers of T cells secreting interferon-gamma (IFN-γ), more IFN-γ in splenocyte culture supernatant, more Th1 and Tc1 cells, and higher ratios of Th1/Th2 and Tc1/Tc2 cells in whole blood, indicating a predominant Th1 immune response to treatment. Infected mice treated with doses of 100 μg Ag85A/B chimeric DNA had an extended time until death of 50% of the animals that was markedly longer than the saline and vector control groups, and the death rate at 1 month after the last dose was lower than that in the other groups. Compared with the saline group, 100 μg Ag85A/B chimeric DNA and 100 μg Ag85A DNA reduced the pulmonary bacterial loads by 0.79 and 0.45 logs, and the liver bacterial loads by 0.52 and 0.50 logs, respectively. Pathological changes in the lungs were less, and the lesions were more limited. These results show that Ag85A/B chimeric DNA was effective for the treatment of TB, significantly increasing the cellular immune response and inhibiting the growth of M. tuberculosis.