Research Article: Manganese acquisition is essential for virulence of Enterococcus faecalis

Date Published: September 20, 2018

Publisher: Public Library of Science

Author(s): Cristina Colomer-Winter, Ana L. Flores-Mireles, Shannon P. Baker, Kristi L. Frank, Aaron J. L. Lynch, Scott J. Hultgren, Todd Kitten, José A. Lemos, Eric P. Skaar.


Manganese (Mn) is an essential micronutrient that is not readily available to pathogens during infection due to an active host defense mechanism known as nutritional immunity. To overcome this nutrient restriction, bacteria utilize high-affinity transporters that allow them to compete with host metal-binding proteins. Despite the established role of Mn in bacterial pathogenesis, little is known about the relevance of Mn in the pathophysiology of E. faecalis. Here, we identified and characterized the major Mn acquisition systems of E. faecalis. We discovered that the ABC-type permease EfaCBA and two Nramp-type transporters, named MntH1 and MntH2, work collectively to promote cell growth under Mn-restricted conditions. The simultaneous inactivation of EfaCBA, MntH1 and MntH2 (ΔefaΔmntH1ΔmntH2 strain) led to drastic reductions (>95%) in cellular Mn content, severe growth defects in body fluids (serum and urine) ex vivo, significant loss of virulence in Galleria mellonella, and virtually complete loss of virulence in rabbit endocarditis and murine catheter-associated urinary tract infection (CAUTI) models. Despite the functional redundancy of EfaCBA, MntH1 and MntH2 under in vitro or ex vivo conditions and in the invertebrate model, dual inactivation of efaCBA and mntH2 (ΔefaΔmntH2 strain) was sufficient to prompt maximal sensitivity to calprotectin, a Mn- and Zn-chelating host antimicrobial protein, and for the loss of virulence in mammalian models. Interestingly, EfaCBA appears to play a prominent role during systemic infection, whereas MntH2 was more important during CAUTI. The different roles of EfaCBA and MntH2 in these sites could be attributed, at least in part, to the differential expression of efaA and mntH2 in cells isolated from hearts or from bladders. Collectively, this study demonstrates that Mn acquisition is essential for the pathogenesis of E. faecalis and validates Mn uptake systems as promising targets for the development of new antimicrobials.

Partial Text

While normal residents of the gastrointestinal (GI) tract of animals and humans, enterococci are also the third most frequent cause of hospital-acquired infections and a major threat to public health due to the alarming rise of multidrug-resistant isolates [1]. Enterococcal infections in humans are mainly caused by Enterococcus faecalis and Enterococcus faecium, with the great majority of infections (~70%) caused by E. faecalis. Collectively, enterococci rank as the third leading etiological agent in infective endocarditis (IE) [2], second in complicated urinary tract infections (UTI) [3], and one of the leading causes of device-associated infections and bacteremia [1]. Despite the recent introduction of new antibiotics active against both E. faecalis and E. faecium (i.e. daptomycin, linezolid, tigecycline), the indiscriminate use of antibiotics and the rise in elderly and severely ill populations susceptible to infection continues to contribute to a worldwide increase in enterococcal infections [4, 5]. The pathogenic potential of E. faecalis, and more generally, of all enterococci, has been largely attributed to their harsh and extremely durable nature, which includes intrinsic tolerance to commonly-used antibiotics (such as cephalosporins), chlorine, alcohol-based detergents, and an ability to survive extreme fluctuations in temperature, pH, oxygen tension, humidity and nutrient availability [1].

In this study we confirm previous in silico predictions that the E. faecalis core genome encodes three bona fide Mn transporters: one ABC-type (EfaCBA) and two Nramp-type transporters (MntH1 and MntH2) [27]. Early studies showed that the virulence of an E. faecalis efaA mutant was slightly delayed in a mouse peritonitis model despite the fact that the strain failed to display noticeable phenotypes in vitro [33, 36]. Here, we provide an explanation for such findings by showing that only the simultaneous inactivation of two or all three transporters can drastically impair Mn homeostasis under laboratory as well as in vivo conditions. Specifically, the ΔefaΔmntH1ΔmntH2 triple mutant strain was unable to grow (or grew very poorly) in Mn-restricted environments—a condition commonly encountered in human tissues—and was unable to robustly infect vertebrate and invertebrate animal hosts. To our knowledge, this is the first description of an E. faecalis mutant strain being virtually avirulent in multiple animal infection models. Moreover, this is also the first demonstration that the ability to acquire Mn using high affinity transporters within the urinary tract environment was shown to be an important aspect for the development of bacterial UTI.