SummaryDiscriminating between commensal and pathogenic states of opportunistic pathogens is critical for host mucosal defense and homeostasis. The opportunistic human fungal pathogen Candida albicans is also a constituent of the normal oral flora and grows either as yeasts or hyphae. We demonstrate that oral epithelial cells orchestrate an innate response to C. albicans via NF-κB and a biphasic MAPK response. Activation of NF-κB and the first MAPK phase, constituting c-Jun activation, is independent of morphology and due to fungal cell wall recognition. Activation of the second MAPK phase, constituting MKP1 and c-Fos activation, is dependent upon hypha formation and fungal burdens and correlates with proinflammatory responses. Such biphasic response may allow epithelial tissues to remain quiescent under low fungal burdens while responding specifically and strongly to damage-inducing hyphae when burdens increase. MAPK/MKP1/c-Fos activation may represent a “danger response” pathway that is critical for identifying and responding to the pathogenic switch of commensal microbes.

A comparison has been made between manual and automated DNA sequencing procedures to evaluate the ability to distinguish mixtures of wild-type and mutant sequences. Quantitative detection of such mixtures of HIV-1 drug resistance mutations was best achieved using an automated system that uses fluorescent-labelled sequencing primers. This procedure has a wide range of applications in clinical research, including heterozygote analysis. Software that automatically reports mixed-base positions is presented.

Mutations can accumulate in the protease and gag genes of human immunodeficiency virus in patients who fail therapy with protease inhibitor drugs. Mutations within protease, the drug target, have been extensively studied. Mutations in gag have been less well studied, mostly concentrating on cleavage sites. A retroviral vector system has been adapted to study full-length gag, protease, and reverse transcriptase genes from patient-derived viruses. Patient plasma-derived mutant full-length gag, protease, and gag-protease from a multidrug-resistant virus were studied. Mutant protease alone led to a 95% drop in replication capacity that was completely rescued by coexpressing the full-length coevolved mutant gag gene. Cleavage site mutations have been shown to improve the replication capacity of mutated protease. Strikingly, in this study, the matrix region and part of the capsid region from the coevolved mutant gag gene were sufficient to achieve full recovery of replication capacity due to the mutant protease, without cleavage site mutations. The same region of gag from a second, unrelated, multidrug-resistant clinical isolate also rescued the replication capacity of the original mutant protease, suggesting a common mechanism that evolves with resistance to protease inhibitors. Mutant gag alone conferred reduced susceptibility to all protease inhibitors and acted synergistically when linked to mutant protease. The matrix region and partial capsid region of gag sufficient to rescue replication capacity also conferred resistance to protease inhibitors. Thus, the amino terminus of Gag has a previously unidentified and important function in protease inhibitor susceptibility and replication capacity.The development of antiretroviral drugs and their use in highly active antiretroviral therapy have led to the ability to effectively control human immunodeficiency virus (HIV) replication in infected patients. The emergence of resistance to these drugs, while reduced when the drugs are used in combination, remains a problem. A recent study has estimated that by 10 years of treatment, nearly 10% of patients will have experienced resistance to the three main classes of drugs that form the basis of highly active antiretroviral therapy. Further, the risk of death within 5 years after extensive triple-class failure is 10% (31). Resistance develops due to several factors, including the high error rate of reverse transcriptase (RT), viral recombination, high viral turnover, and suboptimal drug levels in patients. Mutations emerging in viruses can lead to cross-class resistance.Resistance to protease inhibitors (PI) occurs by the stepwise accumulation of mutations in protease itself, many of which lead to a reduced replication capacity (RC) of the virus (5-7, 22, 23, 37). Primary, or major, mutations mostly lead to a reduced affinity for the inhibitor through alterations in the enzyme active site. Further secondary, or minor, mutations also arise in protease that have little effect on inhibitor binding and therefore do not convey res...

Oral epithelial cells detect the human pathogenic fungus Candida albicans via NF-κB and a bi-phasic mitogen-activated protein kinase (MAPK) signaling response. However, discrimination between C. albicans yeast and hyphal forms is mediated only by the MAPK pathway, which constitutes activation of the MAPK phosphatase MKP1 and the c-Fos transcription factor and is targeted against the hyphal form. Given that C. albicans is not the only Candida species capable of filamentation or causing mucosal infections, we sought to determine whether this MAPK/MKP1/c-Fos mediated response mechanism was activated by other pathogenic Candida species, including C. dubliniensis, C. tropicalis, C. parapsilosis, C. glabrata and C. krusei. Although all Candida species activated the NF-κB signaling pathway, only C. albicans and C. dubliniensis were capable of inducing MKP1 and c-Fos activation, which directly correlated with hypha formation. However, only C. albicans strongly induced cytokine production (G-CSF, GM-CSF, IL-6 and IL-1α) and cell damage. Candida dubliniensis, C. tropicalis and C. parapsilosis were also capable of inducing IL-1α and this correlated with mild cell damage and was dependent upon fungal burdens. Our data demonstrate that activation of the MAPK/MKP1/c-Fos pathway in oral epithelial cells is specific to C. dubliniensis and C. albicans hyphae.

Our findings demonstrate the importance of considering protease inhibitor susceptibility in the context of full-length gag, particularly with respect to the range of HIV-1 subtypes circulating worldwide.