The Host Immune System Facilitates Disseminated Staphylococcus aureus Disease Due to Phagocytic Attraction to Candida albicans during Coinfection: a Case of Bait and Switch

Abstract

Invasive Staphylococcus aureus infections account for 15 to 50% of fatal bloodstream infections annually. These disseminated infections often arise without a defined portal of entry into the host but cause high rates of mortality. The fungus Candida albicans and the Gram-positive bacterium S. aureus can form polymicrobial biofilms on epithelial tissue, facilitated by the C. albicans adhesin encoded by ALS3 While a bacterium-fungus interaction is required for systemic infection, the mechanism by which bacteria disseminate from the epithelium to internal organs is unclear. In this study, we show that highly immunogenic C. albicans hyphae attract phagocytic cells, which rapidly engulf adherent S. aureus and subsequently migrate to cervical lymph nodes. Following S. aureus-loaded phagocyte translocation from the mucosal surface, S. aureus produces systemic disease with accompanying morbidity and mortality. Our results suggest a novel role for the host in facilitating a bacterium-fungus infectious synergy, leading to disseminated staphylococcal disease.

Keywords: Candida albicans; Staphylococcus aureus; biofilms; innate immunity; polymicrobial.

FIG 1

S. aureus cells remain adherent to a single point on C. albicans hyphae in coculture in vitro. (A) Time-lapse microscopy was used to visualize S. aureus adhering to growing hyphae of C. albicans. (B) Active phagocytosis of adherent S. aureus from the hyphae of C. albicans was monitored for over 1 h after the introduction of macrophages. Macrophages can be seen extracting S. aureus from distant areas (top series) or crawling along hypha and removing nearby S. aureus (bottom series). (C) The impact of serum on immune cell targeting was tested by coating with 10% serum prior to macrophage application. In total, macrophages preferentially attached to C. albicans hyphae (center, black bars) over PS (center, gray bars), and after 6 h, 90% of all macrophages were attached to hyphae, where phagocytic activity occurred. Within individual views, an average of 25 macrophages (Mf) could be seen attaching to hyphae (right, black dots) and only a select few were detected still attached to PS (right, open squares). (D) The absence of serum did not impact the phagocytosis of S. aureus on the hyphae of C. albicans. Actively phagocytosing macrophages were scored relative to their hypha-associated state (center, black bars, and right, black dots) or surface-associated state (center, gray bars, and right, open squares). Bars = 20 μm. P values were determined by Student's t test. **, P <0.001; ***, P < 0.0001.

FIG 2

In vivo oral coinfection demonstrates trafficking of phagocytes from tongue tissue to draining cervical lymph nodes and progression of systemic MRSA infection. (A and B) Numbers of CFU in tongue tissue following administration of Hanks balanced salt solution (HBSS), Candida albicans SC5314 alone (CA), methicillin-resistant Staphylococcus aureus USA300 alone (SA), or Candida albicans SC5314 alone and methicillin-resistant Staphylococcus aureus USA300 (CA+SA) to the oral cavity of C57BL/6 mice at 1 to 2 days (B) or 3 days (A) following oral inoculation of S. aureus. (C and D) Numbers of CFU in the draining cervical lymph nodes (C) and kidneys (D) at 3 days post-oral inoculation with S. aureus. (E) Flow cytometry analysis of cervical lymph node (LN) cell suspensions following fixation, host macrophage (PE-Cy7A-F4/80) or neutrophil (Pacific Blue [PAC Blue]-Ly6G) antibody labeling, permeabilization, and methicillin-resistant S. aureus (FITC-S. aureus antibody [Ab]) labeling 3 days following oral inoculation of uninfected controls or S. aureus and C. albicans dual species-infected mice. (F and G) Numbers of CFU in cervical lymph nodes (F) and kidneys (G) at 1 to 2 days postinoculation of S. aureus. P values were determined by one-way analysis of variance with Dunnett’s multiple-comparison test. *, P < 0.05; **, P < 0.001.

FIG 3

Innate immune cells enter tongue tissue to colocalize at hyphae and bacteria within 1 day of coinfection. Tongues were excised from immunocompromised animals, embedded in paraffin, and stained with PAS. Images represent tongues from two uninfected mice (A and B) and two infected mice (C and D) at 1 day after coinfection with S. aureus and C. albicans. Red arrows indicate PMN invasion up hyphae (D), and red boxes indicate staphylococci clumping tightly with C. albicans hyphae and cells (E) or monocytes surrounding hyphae to seize bacteria (F; the red box and the red box with a red arrow indicate ingested staphylococci). Magnifications, ×5 (A and B), ×20 (C and D), and ×40 (E and F). (G) Immunohistochemically labeled macrophages (red filter) were seen engulfing S. aureus (green filter) only in coinfected mice. The fluorescent images were counterstained with DAPI to show nuclei (blue filter). An orthogonal image (bottom right) shows S. aureus within a macrophage. Magnifications, ×100.