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1.  Oral and Vaginal Epithelial Cell Lines Bind and Transfer Cell-Free Infectious HIV-1 to Permissive Cells but Are Not Productively Infected 
PLoS ONE  2014;9(5):e98077.
The majority of HIV-1 infections worldwide are acquired via mucosal surfaces. However, unlike the vaginal mucosa, the issue of whether the oral mucosa can act as a portal of entry for HIV-1 infection remains controversial. To address potential differences with regard to the fate of HIV-1 after exposure to oral and vaginal epithelium, we utilized two epithelial cell lines representative of buccal (TR146) and pharyngeal (FaDu) sites of the oral cavity and compared them with a cell line derived from vaginal epithelium (A431) in order to determine (i) HIV-1 receptor gene and protein expression, (ii) whether HIV-1 genome integration into epithelial cells occurs, (iii) whether productive viral infection ensues, and (iv) whether infectious virus can be transferred to permissive cells. Using flow cytometry to measure captured virus by HIV-1 gp120 protein detection and western blot to detect HIV-1 p24 gag protein, we demonstrate that buccal, pharyngeal and vaginal epithelial cells capture CXCR4- and CCR5-utilising virus, probably via non-canonical receptors. Both oral and vaginal epithelial cells are able to transfer infectious virus to permissive cells either directly through cell-cell attachment or via transcytosis of HIV-1 across epithelial cells. However, HIV-1 integration, as measured by real-time PCR and presence of early gene mRNA transcripts and de novo protein production were not detected in either epithelial cell type. Importantly, both oral and vaginal epithelial cells were able to support integration and productive infection if HIV-1 entered via the endocytic pathway driven by VSV-G. Our data demonstrate that under normal conditions productive HIV-1 infection of epithelial cells leading to progeny virion production is unlikely, but that epithelial cells can act as mediators of systemic viral dissemination through attachment and transfer of HIV-1 to permissive cells.
doi:10.1371/journal.pone.0098077
PMCID: PMC4032250  PMID: 24857971
2.  Candida albicans Cell Wall Glycosylation May Be Indirectly Required for Activation of Epithelial Cell Proinflammatory Responses ▿  
Infection and Immunity  2011;79(12):4902-4911.
Oral epithelial cells discriminate between the yeast and hyphal forms of Candida albicans via the mitogen-activated protein kinase (MAPK) signaling pathway. This occurs through phosphorylation of the MAPK phosphatase MKP1 and activation of the c-Fos transcription factor by the hyphal form. Given that fungal cell wall polysaccharides are critical in host recognition and immune activation in myeloid cells, we sought to determine whether β-glucan and N- or O-glycosylation was important in activating the MAPK/MKP1/c-Fos hypha-mediated response mechanism and proinflammatory cytokines in oral epithelial cells. Using a series of β-glucan and N- and O-mannan mutants, we found that N-mannosylation (via Δoch1 and Δpmr1 mutants) and O-mannosylation (via Δpmt1 and Δmnt1 Δmnt2 mutants), but not phosphomannan (via a Δmnn4 mutant) or β-1,2 mannosylation (via Δbmt1 to Δbmt6 mutants), were required for MKP1/c-Fos activation, proinflammatory cytokine production, and cell damage induction. However, the N- and O-mannan mutants showed reduced adhesion or lack of initial hypha formation at 2 h, resulting in little MKP1/c-Fos activation, or restricted hypha formation/pseudohyphal formation at 24 h, resulting in minimal proinflammatory cytokine production and cell damage. Further, the α-1,6-mannose backbone of the N-linked outer chain (corresponding to a Δmnn9 mutant) may be required for epithelial adhesion, while the α-1,2-mannose component of phospholipomannan (corresponding to a Δmit1 mutant) may contribute to epithelial cell damage. β-Glucan appeared to play no role in adhesion, epithelial activation, or cell damage. In summary, N- and O-mannosylation defects affect the ability of C. albicans to induce proinflammatory cytokines and damage in oral epithelial cells, but this may be due to indirect effects on fungal pathogenicity rather than mannose residues being direct activators of the MAPK/MKP1/c-Fos hypha-mediated immune response.
doi:10.1128/IAI.05591-11
PMCID: PMC3232641  PMID: 21930756
3.  Evaluation of the Role of Candida albicans Agglutinin-Like Sequence (Als) Proteins in Human Oral Epithelial Cell Interactions 
PLoS ONE  2012;7(3):e33362.
The fungus C. albicans uses adhesins to interact with human epithelial surfaces in the processes of colonization and pathogenesis. The C. albicans ALS (agglutinin-like sequence) gene family encodes eight large cell-surface glycoproteins (Als1-Als7 and Als9) that have adhesive function. This study utilized C. albicans Δals mutant strains to investigate the role of the Als family in oral epithelial cell adhesion and damage, cytokine induction and activation of a MAPK-based (MKP1/c-Fos) signaling pathway that discriminates between yeast and hyphae. Of the eight Δals mutants tested, only the Δals3 strain showed significant reductions in oral epithelial cell adhesion and damage, and cytokine production. High fungal:epithelial cell multiplicities of infection were able to rescue the cell damage and cytokine production phenotypes, demonstrating the importance of fungal burden in mucosal infections. Despite its adhesion, damage and cytokine induction phenotypes, the Δals3 strain induced MKP1 phosphorylation and c-Fos production to a similar extent as control cells. Our data demonstrate that Als3 is involved directly in epithelial adhesion but indirectly in cell damage and cytokine induction, and is not the factor targeted by oral epithelial cells to discriminate between the yeast and hyphal form of C. albicans.
doi:10.1371/journal.pone.0033362
PMCID: PMC3299778  PMID: 22428031
4.  Activation of MAPK/c-Fos induced responses in oral epithelial cells is specific to Candida albicans and Candida dubliniensis hyphae 
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.
doi:10.1007/s00430-011-0209-y
PMCID: PMC3257392  PMID: 21706283
Candida albicans; Candida dubliniensis; Hypha formation; MAPK; MKP1; c-Fos; NF-κB; Oral epithelium; Innate immunity
5.  Candida albicans Yeast and Hyphae are Discriminated by MAPK Signaling in Vaginal Epithelial Cells 
PLoS ONE  2011;6(11):e26580.
We previously reported that a bi-phasic innate immune MAPK response, constituting activation of the mitogen-activated protein kinase (MAPK) phosphatase MKP1 and c-Fos transcription factor, discriminates between the yeast and hyphal forms of Candida albicans in oral epithelial cells (ECs). Since the vast majority of mucosal Candida infections are vaginal, we sought to determine whether a similar bi-phasic MAPK-based immune response was activated by C. albicans in vaginal ECs. Here, we demonstrate that vaginal ECs orchestrate an innate response to C. albicans via NF-κB and MAPK signaling pathways. However, unlike in oral ECs, the first MAPK response, defined by c-Jun transcription factor activation, is delayed until 2 h in vaginal ECs but is still independent of hypha formation. The ‘second’ or ‘late’ MAPK response, constituting MKP1 and c-Fos transcription factor activation, is identical to oral ECs and is dependent upon both hypha formation and fungal burdens. NF-κB activation is immediate but independent of morphology. Furthermore, the proinflammatory response in vaginal ECs is different to oral ECs, with an absence of G-CSF and CCL20 and low level IL-6 production. Therefore, differences exist in how C. albicans activates signaling mechanisms in oral and vaginal ECs; however, the activation of MAPK-based pathways that discriminate between yeast and hyphal forms is retained between these mucosal sites. We conclude that this MAPK-based signaling pathway is a common mechanism enabling different human epithelial tissues to orchestrate innate immune responses specifically against C. albicans hyphae.
doi:10.1371/journal.pone.0026580
PMCID: PMC3210759  PMID: 22087232
6.  MAPK, MKP1 and c-Fos Discriminate Candida albicans Yeast from Hyphae in Epithelial Cells 
Cell host & microbe  2010;8(3):225-235.
SUMMARY
Host mechanisms enabling discrimination between the commensal and pathogenic states of opportunistic pathogens are critical in mucosal defense and homeostasis. Here, we demonstrate that oral epithelial cells orchestrate an innate response to the human fungal pathogen Candida albicans via NF-κB and a bi-phasic MAPK response. Activation of NF-κB and the first MAPK phase, constituting c-Jun activation, is independent of morphology and due to the recognition of fungal cell wall structures. 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. This MAPK-based discriminatory pathway may provide a mechanism for epithelial tissues to remain quiescent in the presence of low fungal burdens whilst responding specifically and strongly to damage-inducing hyphae when burdens increase. MAPK/MKP1/c-Fos activation may thus comprise a `danger response' pathway in vivo and may be critical in identifying when this normally commensal fungus has become pathogenic.
doi:10.1016/j.chom.2010.08.002
PMCID: PMC2991069  PMID: 20833374
7.  A Biphasic Innate Immune MAPK Response Discriminates between the Yeast and Hyphal Forms of Candida albicans in Epithelial Cells 
Cell Host & Microbe  2010;8(3):225-235.
Summary
Discriminating 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.
Highlights
► NF-κB and MAPK control epithelial effector responses against Candida albicans ► c-Jun activation is independent of morphology and due to fungal cell wall recognition ► MAPK/MKP-1/c-Fos pathway activation is dependent on fungal hyphae and burdens ► MAPK discriminatory response may dictate C. albicans mucosal colonization in vivo
doi:10.1016/j.chom.2010.08.002
PMCID: PMC2991069  PMID: 20833374

Results 1-7 (7)