Pathogenic mechanisms of Candida glabrata in oral candidiasis, especially because of its inability to form hyphae, are understudied. Since both Candida albicans and C. glabrata are frequently co-isolated in oropharyngeal candidiasis (OPC), we examined their co-adhesion in vitro and observed adhesion of C. glabrata only to C. albicans hyphae microscopically. Mice were infected sublingually with C. albicans or C. glabrata individually, or with both species concurrently, to study their ability to cause OPC. Infection with C. glabrata alone resulted in negligible infection of tongues; however, colonization by C. glabrata was increased by co-infection or a pre-established infection with C. albicans. Furthermore, C. glabrata required C. albicans for colonization of tongues, since decreasing C. albicans burden with fluconazole also reduced C. glabrata. C. albicans hyphal wall adhesins Als1 and Als3 were important for in vitro adhesion of C. glabrata and to establish OPC. C. glabrata cell wall protein coding genes EPA8, EPA19, AWP2, AWP7, and CAGL0F00181 were implicated in mediating adhesion to C. albicans hyphae and remarkably, their expression was induced by incubation with germinated C. albicans. Thus, we found a near essential requirement for the presence of C. albicans for both initial colonization and establishment of OPC infection by C. glabrata.

Sjögren's syndrome is a chronic autoimmune disorder characterized by inflammation of salivary glands resulting in impaired secretory function. Our present studies indicate that chronic exposure of salivary epithelium to TNF-α and/or IFN-γ alters tight junction integrity, leading to secretory dysfunction. Resolvins of the D-series (RvDs) are endogenous lipid mediators derived from DHA that regulate excessive inflammatory responses leading to resolution and tissue homeostasis. In this study, we addressed the hypothesis that activation of the RvD1 receptor ALX/FPR2 in salivary epithelium prevents and/or resolves the TNF-α-mediated disruption of acinar organization and enhances monolayer formation. Our results indicate that 1) the RvD1 receptor ALX/FPR2 is present in fresh, isolated cells from mouse salivary glands and in cell lines of salivary origin; and 2) the agonist RvD1 (100 ng/ml) abolished tight junction and cytoskeletal disruption caused by TNF-α and enhanced cell migration and polarity in salivary epithelium. These effects were blocked by the ALX/FPR2 antagonist butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe. The ALX/FPR2 receptor signals via modulation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways since, in our study, blocking PI3K activation with LY294002, a potent and selective PI3K inhibitor, prevented RvD1-induced cell migration. Furthermore, Akt gene silencing with the corresponding siRNA almost completely blocked the ability of Par-C10 cells to migrate. Our findings suggest that RvD1 receptor activation promotes resolution of inflammation and tissue repair in salivary epithelium, which may have relevance in the restoration of salivary gland dysfunction associated with Sjögren's syndrome.

Nelson JW, Leigh NJ, Mellas RE, McCall AD, Aguirre A, Baker OJ. ALX/FPR2 receptor for RvD1 is expressed and functional in salivary glands.

The opportunistic fungal pathogen Candida albicans is capable of adhering to the oral mucosa despite forces created by salivary flow. Although many fungal adhesion proteins have been identified, less is known about the temporal development of cell adhesion and biofilm growth in a flow environment. In this study, we use a flow system with real-time imaging of C. albicans cells as they adhere and grow. Rates of cell attachment and dispersion of C. albicans knockout strains of putative adhesins, transcription factors, and deletions with a hyperfilamentous phenotype were quantified during 18 h of biofilm development. Cell adhesion under flow is a multi-phase process initiated with cell rolling, then an initial firm attachment to the substrate occurs. After attachment, cells enter a growth phase where cells either commit to adherence or disperse. C. albicans Δ eap1 , Δ hwp2 , Δ hyr1 , and Δ ihd1 cells had significantly reduced initial attachment and subsequent adhesion, while Δ als1 /Δ als3 had no change in initial attachment but reduced adhesion maintenance. WT cells had increased adhesion during the late growth phase when hyphae were more highly expressed. Hyperfilamentous strains had 10-fold higher total biofilm growth, a result of significantly reduced detachment rates, showing that hyphal morphogenesis is important for adhesion maintenance in the developing biofilm. The rate of C. albicans biomass dispersion was most important for determining the density of the mature biomass. Adhesion maintenance was mediated in part by Ywp1, a protein previously thought to regulate dispersion, thus it functions as an adhesion maintenance protein in C. albicans .

ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanni, Pseudomonas aeruginosa, and Enterobacter species)pathogens have characteristic multiple-drug resistance and cause an increasing number of nosocomial infections worldwide. Peptide-based therapeutics to treat ESKAPE infections might be an alternative to conventional antibiotics. Histatin 5 (Hst 5) is a salivary cationic histidine-rich peptide produced only in humans and higher primates. It has high antifungal activity against Candida albicans through an energy-dependent, non-lytic process; but its bactericidal effects are less known. We found Hst 5 has bactericidal activity against S. aureus (60-70% killing) and A. baumannii (85-90% killing) in 10 and 100 mM sodium phosphate buffer (NaPB), while killing of >99% of P. aeruginosa, 60-80% E. cloacae and 20-60% of E. faecium was found in 10 mM NaPB. Hst 5 killed 60% of biofilm cells of P. aeruginosa, but had reduced activity against biofilms of S. aureus and A. baumannii. Hst 5 killed 20% of K. pneumonia biofilm cells but not planktonic cells. Binding and uptake studies using FITC-labeled Hst 5 showed E. faecium and E. cloacae killing required Hst 5 internalization and was energy dependent, while bactericidal activity was rapid against P. aeruginosa and A. baumannii suggesting membrane disruption. Hst 5-mediated killing of S. aureus was both non-lytic and energy independent. Additionally, we found that spermidine conjugated Hst 5 (Hst5-Spd) had improved killing activity against E. faecium, E. cloacae, and A. baumannii. Hst 5 or its derivative has antibacterial activity against five out of six ESKAPE pathogens and may be an alternative treatment for these infections.

is a newly identified species causing invasive candidemia and candidiasis. It has broad multidrug resistance (MDR) not observed for other pathogenic species. Histatin 5 (Hst 5) is a well-studied salivary cationic peptide with significant antifungal activity against and is an attractive candidate for treating MDR fungi, since antimicrobial peptides induce minimal drug resistance. We investigated the susceptibility of to Hst 5 and neutrophils, two first-line innate defenses in the human host. The majority of clinical isolates, including fluconazole-resistant strains, were highly sensitive to Hst 5: 55 to 90% of cells were killed by use of 7.5 μM Hst 5. Hst 5 was translocated to the cytosol and vacuole in cells; such translocation is required for the killing of by Hst 5. The inverse relationship between fluconazole resistance and Hst 5 killing suggests different cellular targets for Hst 5 than for fluconazole. showed higher tolerance to oxidative stress than, and higher survival within neutrophils, which correlated with resistance to oxidative stress Thus, resistance to reactive oxygen species (ROS) is likely one, though not the only, important factor in the killing of by neutrophils. Hst 5 has broad and potent candidacidal activity, enabling it to combat MDR strains effectively.

Salivary gland cell differentiation has been a recurring challenge for researchers as primary salivary cells show a loss of phenotype in culture. Particularly, parotid cells show a marked decrease in amylase expression, the loss of tight junction organization and proper cell function. Previously, Matrigel has been used successfully as an extracellular matrix; however, it is not practical for in vivo applications as it is tumorigenic. An alternative method could rely on the use of fibrin hydrogel (FH), which has been used extensively in biomedical engineering applications ranging from cardiovascular tissue engineering to wound-healing experiments. Although several groups have examined the effects of a three-dimensional (3D) environment on salivary cell cultures, little is known about the effects of FH on salivary cell cultures. The current study developed a 3D cell culture model to support parotid gland cell differentiation using a combination of FH and growth factor-reduced Matrigel (GFR-MG). Furthermore, FH polymerized with a combination of EGF and IGF-1 induced formation of 3D spheroids capable of amylase expression and an agonist-induced increase in the intracellular Ca 2 + concentration ([Ca 2 + ] i ) in salivary cells. These studies represent an initial step toward the construction of an artificial salivary gland to restore salivary gland dysfunction. This is necessary to reduce xerostomia in patients with compromised salivary function.

Candida albicans is an opportunistic fungal pathogen that can infect oral mucosal surfaces while being under continuous flow from saliva. Under specific conditions, C. albicans will form microcolonies that more closely resemble the biofilms formed in vivo than standard in vitro biofilm models. However, very little is known about these microcolonies, particularly genomic differences between these specialized biofilm structures and the traditional in vitro biofilms. In this study, we used a novel flow system, in which C. albicans spontaneously forms microcolonies, to further characterize the architecture of fungal microcolonies and their genomics compared to non-microcolony conditions. Fungal microcolonies arose from radially branching filamentous hyphae that increasingly intertwined with one another to form extremely dense biofilms, and closely resembled the architecture of in vivo oropharyngeal candidiasis. We identified 20 core microcolony genes that were differentially regulated in flow-induced microcolonies using RNA-seq. These genes included HWP1, ECE1, IHD1, PLB1, HYR1, PGA10, and SAP5. A predictive algorithm was utilized to identify ten transcriptional regulators potentially involved in microcolony formation. Of these transcription factors, we found that Rob1, Ndt80, Sfl1 and Sfl2, played a key role in microcolony formation under both flow and static conditions and to epithelial surfaces. Expression of core microcolony genes were highly up-regulated in Δsfl1 cells and down-regulated in both Δsfl2 and Δrob1 strains. Microcolonies formed on oral epithelium using C. albicans Δsfl1, Δsfl2 and Δrob1 deletion strains all had altered adhesion, invasion and cytotoxicity. Furthermore, epithelial cells infected with deletion mutants had reduced (SFL2, NDT80, and ROB1) or enhanced (SFL2) immune responses, evidenced by phosphorylation of MKP1 and c-Fos activation, key signal transducers in the hyphal invasion response. This profile of microcolony transcriptional regulators more closely reflects Sfl1 and Sfl2 hyphal regulatory networks than static biofilm regulatory networks, suggesting that microcolonies are a specialized pathogenic form of biofilm.