Journal of Applied Microbiology ISSN 1364-5072


Oral lactoferrin protects against experimental candidiasis in mice K. Velliyagounder, W. Alsaedi, W. Alabdulmohsen, K. Markowitz and D.H. Fine RUTGERS School of Dental Medicine, Newark, NJ, USA

Keywords Candida albicans, experimental candidiasis, human lactoferrin, lactoferrin knockout, oral candidiasis. Correspondence Kabilan Velliyagounder, Department of Oral Biology, RUTGERS School of Dental Medicine, 185 South Orange Ave, Newark, NJ 07103, USA. E-mail: [email protected] 2014/1265: received 20 June 2014, revised 2 October 2014 and accepted 14 October 2014 doi:10.1111/jam.12666

Abstract Aims: To determine the role of human lactoferrin (hLF) in protecting the oral cavities of mice against Candida albicans infection in lactoferrin knockout (LFKO / ) mice was compared to wild-type (WT) mice. We also aim to determine the protective role of hLF in LFKO / mice. Methods and Results: Antibiotic-treated immunosuppressed mice were inoculated with C. albicans (or sham infection) by oral swab and evaluated for the severity of infection after 7 days of infection. To determine the protective role of hLF, we added 03% solution of hLF to the drinking water given to some of the mice. CFU count, scoring of lesions and microscopic observations were carried out to determine the severity of infection. LFKO / I mice showed a 2 log (P = 0001) higher CFUs of C. albicans in the oral cavity compared to the WT mice infected with C. albicans (WTI). LFKO / I mice given hLF had a 3 log (P = 0001) reduction in CFUs in the oral cavity compared to untreated LFKO / I mice. The severity of infection, observed by light microscopy, revealed that the tongue of the LFKO / I mice showed more white patches compared to WTI and LFKO / I + hLF mice. Scanning electron microscopic observations revealed that more filiform papillae were destroyed in LFKO / I mice when compared to WTI or LFKO / I + hLF mice. Conclusions: Human LF is important in protecting mice from oral C. albicans infection. Administered hLF may be used to prevent C. albicans infection. Significance and Impact of the Study: Human LF, a multifunctional ironbinding glycoprotein can be used as a therapeutic active ingredient in oral healthcare products against C. albicans.

Introduction Candida albicans, the most prevalent fungal biofilm forming pathogen, is responsible for several types of oral and systemic infections, including oropharyngeal candidiasis (OPC) or thrush, denture stomatitis (DS) and bloodborne candidemia infections (Drobacheff et al. 1996; Cannon and Chaffin 1999; Ruhnke 2006). In the United States, candidemia is the fourth most common bloodstream infection seen in hospitalized patients (Edmond et al. 1999). Sixty to ninety per cent of HIV-infected patients are susceptible to fungal infections, specifically OPC (Moore and Chaisson 1996). Candida albicans bloodstream infections are polymicrobial and often associated with a variety of pathogenic bacterial species, 212

including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis (Morales and Hogan 2010; Peleg et al. 2010; Kovac et al. 2013). The mortality rate of systemic Candida infection was reported to be 71–97% (Fraser et al. 1992). Also, it can cause infections in patients with diabetes and those who have a long history of antibiotic and immunosuppressive drug usage. Candida albicans’ main targets are immunocompromised patients, neonates and the elderly, especially denture wearers (Akpan and Morgan 2002). It has been reported that 65% of denture wearers develop Candidaassociated DS (Daniluk et al. 2006). Candida albicans pathogenicity mainly relies on the immune status of the host and the virulence factors of the fungus. There are several virulence factors that are responsible for the

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pathogenicity of C. albicans including host cell adhesion, hydrolytic enzymes production, dimorphic phenotype and the ability to form a biofilm on biotic and abiotic surfaces (Cutler et al. 1991). There are several reports showing resistant strains of C. albicans due to recurrent episodes of OPC and prolonged use of azole antifungal treatment (Ruhnke et al. 1994; White and Goetz 1994; Drobacheff et al. 1996; Hamza et al. 2008). Currently, different antimicrobials and antifungal or a combination of both are being used to treat oral and systemic infections caused by C. albicans. However, being an adherent organism with a tendency to invade tissues, it is extremely difficult to eradicate C. albicans from the oral cavity and other organs. In addition, the emergence of resistant strains makes it difficult to treat C. albicans resulting in a high percentage of recurrence. Frequent use of antifungal drugs not only increases fungal resistance but also increases the susceptibility to unpleasant side effects. Therefore, new strategies are required for the development of antifungal agents and combined treatments against C. albicans infection. Lactoferrin (LF) is an important component of the innate immune system found in high levels in human milk (HM), especially high concentration in the colostrum. It is also freely available in the subgingival environment as a result of neutrophil degranulation (Tomita et al. 2009). LF exhibits bacteriostatic and bactericidal activities against a wide range of Gram-negative and Gram-positive bacteria (Gonzalez-Chavez et al. 2009). The anti-Candidal activity of hLF was first reported by Kirkpatrick et al. (1971) and since then there have been several reports demonstrating the anticandidal activity of hLF in vitro and in vivo (Abe et al. 2000; Samaranayake et al. 2001; Lupetti et al. 2003, 2007; Takakura et al. 2003, 2004; Andres et al. 2008). It has also been reported that hLF prevented Candida species from producing pseudohyphea for 3 days (Al-Sheikh 2009). The mechanism of hLF anticandidal activity against C. albicans may be related to cell surface alterations, leakage of proteins and formation of surface blebs (Nikawa et al. 1993, 1995). A study showed that prophylactic treatment of talactoferrin alfa (TLF) had a significant effect on neonatal rats with C. albicans and S. Staphylococcus epidermidis coinfection (Venkatesh et al. 2007). Another study demonstrated the synergic effect of LF and lysozyme against C. albicans (Samaranayake et al. 2001). Moreover, it has been reported that transgenic porcine LF milk-fed mice were protected against E. coli, Staph. aureus and C. albicans infections of their digestive tract (Yen et al. 2009). Masci (2000) had reported that including LF and lysozyme in a mouthwash was useful in treating refractory

Role of lactoferrin against Candida albicans

oral candidiasis in HIV patients (Masci 2000). Another clinical study also showed that LF in mucoadhesive tablets reduced OPC (Kuipers et al. 2002). There have been many studies demonstrating the protective effect of LF and LF-derived peptides against in vivo infections in mouse models where the animals are capable of producing LF. Candida infection has not been studied in animal models where LF was lacking or where LF deficient mice were given exogenous hLF. We have demonstrated in our previous study that the lactoferrin knockout (LFKO / ) mice had a higher risk and severity of infection with oral bacteria including higher alveolar bone loss with increased expression of proinflammatory cytokines as well as the chemokine expression during infection with Aggregatibacter actinomycetemcomitans (Velusamy et al. 2013). We also reported that IV administration of hLF rapidly cleared A. actinomycetemcomitans and Streptococcus mutans in blood and other organs in LFKO / mice compared to untreated group (Velusamy et al. 2014a,b). Based on our preliminary data and previous studies, we hypothesize that LFKO / mice are more susceptible to C. albicans infections compared to wild-type (WT) mice. It is of our interest to establish a model to examine the use of hLF to control oral candidiasis in the absence of endogenous LF. Therefore, in the present study, we examined experimental oral candidiasis and the eventual patterns of the anticandidal activities in response to hLF treatment in a LFKO / mouse model. This study will provide data necessary to help us understand the use of hLF as an anticandidal agent and its mechanisms of action in vivo in an experimentally induced oral candidiasis mouse model. Materials and methods Candida albicans and growth condition Candida albicans strain (ATCC 90028) obtained from Dr. Gill Diamond (University of Florida, Gainesville, FL) was stored at 80°C in YPD broth containing 05% yeast extract and 10% glycerol until the experiment was performed. The yeast was grown on YPD agar plates at 37°C for 24 h and harvested by microspatula and then suspended in RPMI 1640 medium (Sigma Chemical Co., St. Louis, Mo) containing 25% foetal calf serum. The yeast cell number of the suspension was adjusted to 25 9 107 cells ml 1 using a haemocytometer. In this study, the following chemicals and reagents were used including tetracycline (5 mg ml 1), prednisolone (083 mg ml 1) (Sigma), HM (a generous gift from Dr. Plaut, Tufts-New England Medical Center, Boston, MA) and hLF (Sigma).

Journal of Applied Microbiology 118, 212--221 © 2014 The Society for Applied Microbiology


Role of lactoferrin against Candida albicans

K. Velliyagounder et al.

In vitro effect of HM and hLF on Candida albicans Based on the previous study, LF concentration in HM was adjusted to 200 lg ml 1 as previously described (Plaut et al. 2000). Candida albicans was incubated either with HM or purified LF from HM (200 lg ml 1) for different time points (6, 12, 24, 48 and 72 h) at 37°C. At each time point, the yeast cells were then washed three times with phosphate buffered saline (PBS) (Morrill et al. 2003). The samples were then serially diluted and plated on YPD agar plates, and viable colonies were enumerated after 2 days.

cell suspension (25 9 107 cells ml 1), and the entire oral cavity of the anaesthetized mice was swabbed to produce oral infection. The oral cavities of mice in the control group were swabbed with C. albicans-free media. Human LF administration To determine the protective role of hLF against C. albicans, hLF at a concentration of 03% solution in drinking water (equivalent to 05 g kg 1 day 1) was consecutively administered from 1 day before the infection and throughout the study as previously described (Takakura et al. 2003).

In vivo experimental oral candidiasis Mice: Experimental groups comprised of 7- to 8-weekold male WT (C57BL/6J) and LFKO / mice, a generous gift from Dr. Orla Conneely, (Baylor College of Medicine, Houston, TX, USA). All animal experimental protocols were approved by the campus Institutional Animal Care and Use Committee (IACUC) of Rutgers School of Dental medicine, Newark, NJ, USA. In the murine model of oral candidiasis that was previously developed, oral candidiasis was observed to be nonlethal (Takakura et al. 2003; Wakabayashi et al. 2003). Based on the patterns of infections in these studies, we used six mice for each group and a total of 30 mice were used to test our hypothesis. The following experimental groups were studied: (i) Wild-type control mice (WTC), (ii) Wild-type mice infected with C. albicans (WTI), (iii) LFKO / mice control (LFKO / C), (iv) LFKO / mice infected with C. albicans (LFKO / I) and (v) LFKO / mice infected with C. albicans and treated with hLF (LFKO / I + hLF). Experimental oral candidiasis The animals were randomized, assigned to groups of five and given food and water ad libitum. Candida albicans experimental oral infection was performed according to the published method (Takakura et al. 2003). Tetracycline hydrochloride in drinking water at a concentration of 083 mg ml 1 was given to the mice beginning 1 day before infection and continued throughout the study to reduce oral bacteria. The oral cavity was swabbed before infection and plated on YPD-tetracycline (5 mg ml 1) agar plates to verify the absence of bacteria. Mice were immunosuppressed with two subcutaneous injections of prednisolone at a dose of 100 mg kg 1 of body weight 1 day prior to and 3 days after the infection with C. albicans. Mice were anaesthetized by an intramuscular injection with 50 ll of 2-mg ml 1 chlorpromazine chloride in the thigh. Small cotton bud was soaked in a C. albicans 214

Evaluation of severity of infection Microbiological evaluation The mice in each experimental group were sacrificed with CO2 7 days after C. albicans or sham infection. Microbiological evaluations of the progression of infection were carried out as follows. The whole oral cavity, including the buccal mucosa, the tongue, the soft palate, and other oral mucosal surfaces were swabbed with a cotton bud and placed in a tube containing 5 ml of sterile PBS. The samples were then serially diluted, plated on YPD agar plates with tetracycline incubated at 37°C for 2 days followed by enumeration of CFU of Candida cells (Takakura et al. 2003). Microscopic analysis Light microscopy and SEM. The tongues from the experimental groups were removed, washed with PBS and then photographed using light microscopy (Olympus SZ61, Tokyo, Japan) to observe the severity of the infection. Also, scanning electron microscopic (SEM) analysis was performed to examine morphological changes on the dorsal surfaces of tongues after Candida infection or sham infection. After washing the tongues with PBS, the tongues were fixed with 2% glutaraldehyde for 2 h, dehydrated with different concentrations of ethanol (25, 50, 75, 100%) with each for 25 min and processed for SEM according to a previously published method using an accelerating voltage of 30 kV (Hitachi SEM S2500; Hitachi High Technologies America, Inc., Clarksburg, MD, USA) (Velliyagounder et al. 2012). Macroscopic evaluation Macroscopic evaluation of the infection was indicated by a lesion score from 0 to 4 on the basis of the extent and severity of whitish, curd-like patches on the tongue surface as follows: 0, normal; 1, white patches

Oral lactoferrin protects against experimental candidiasis in mice.

To determine the role of human lactoferrin (hLF) in protecting the oral cavities of mice against Candida albicans infection in lactoferrin knockout (L...
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