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Identification of Mycobacterium avium genes
associated with resistance to host antimicrobial
Nima Motamedi,1Lia Danelishvili2and Luiz E. Bermudez1,2,3
Luiz E. Bermudez
Received 2 January 2014
Accepted 14 May 2014
1Kuzell Institute, California Pacific Medical Center, San Francisco, CA, USA
2Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University,
Corvallis, OR, USA
3Department of Microbiology, College of Science, Oregon State University, OR, USA
Antimicrobial peptides are an important component of the innate immune defence.
Mycobacterium avium subsp. hominissuis (M. avium) is an organism that establishes contact with
the respiratory and gastrointestinal mucosa as a necessary step for infection. M. avium is resistant
to high concentrations of polymyxin B, a surrogate for antimicrobial peptides. To determine gene-
encoding proteins that are associated with this resistance, we screened a transposon library of M.
avium strain 104 for susceptibility to polymyxin B. Ten susceptible mutants were identified and the
inactivated genes sequenced. The great majority of the genes were related to cell wall synthesis
and permeability. The mutants were then examined for their ability to enter macrophages and to
survive macrophage killing. Three clones among the mutants had impaired uptake by
macrophages compared with the WT strain, and all ten clones were attenuated in macrophages.
The mutants were also shown to be susceptible to cathelicidin (LL-37), in contrast to the WT
bacterium. All but one of the mutants were significantly attenuated in mice. In conclusion, this
study indicated that the M. avium envelope is the primary defence against host antimicrobial
Mycobacterium avium subsp. hominissuis (hereafter M.
avium) is a pathogen that infects humans and other
mammals by crossing mucosal barriers. In humans, M.
avium causes disease in both immunocompromised and
immune-competent individuals (Ashitani et al., 2001;
Marras & Daley, 2002). Indication of infection in most of
the patients is connected to signs and symptoms associated
with the respiratory tract or systemic disease (Ashitani et al.,
2001; Marras & Daley, 2002).
In order to cause infection by crossing the respiratory and
intestinal mucosas, M. avium has to resist the action of
antimicrobial peptides present on mucosal surfaces. Human
intestinal mucosa secretes b-defensins, cathelicidin and Reg
IIIb (Bevins & Salzman, 2011; Ouellette, 1999), while chiefly
cathelicidin and defensins are found in the respiratory tract
mucosa (Bals, 2000). Antimicrobial peptides play an impor-
tant role in the host innate response against a number
of bacteria (Sørensen et al., 2008) and perhaps against
High concentrations of antimicrobial peptides are encoun-
tered in the mucus layer, preventing bacteria from moving
closer to the mucosal surface. In the intestinal lumen, M.
avium has to cross two layers of mucus, one of them with
large concentrations of antimicrobial peptides (Johansson
et al., 2011). In fact, work by Johansson’s group has shown
that while the external mucus layer contains many bacteria
and a low concentration of antimicrobial peptides, the
innermost layer of mucus is rich in antimicrobial peptides
and generally deficient in micro-organisms (Johansson
et al., 2008). M. avium does not have flagella or any other
mechanisms to move across the mucus layers towards the
mucosa. Therefore, in order for the bacterium to establish
contact with the mucosal surface, it should possess a
mechanism that would confer resistance to the harmful
environment of the mucus, perhaps for an extended period
of time. We now have evidence that M. avium does not
bind to mucin, which may facilitate the bacterial migration
in the mucus (J. Bechler, K. Gilbert & L. E. Bermudez,
unpublished results). However, the pathogen should also
be able to resist the action of antimicrobial peptides.
Prior studies have suggested that Mycobacterium tuber-
culosis may be susceptible to rabbit and human defensins
and to other antimicrobial peptides (Miyakawa et al.,
Abbreviations: INH, isoniazid; MAC, Mycobacterium avium complex;
OADC, oleic acid, albumin, glucose and catalase; PKS, polyketide
synthase; 1,25 vit D3, 1,25-dihydroxyvitamin D3.
Journal of Medical Microbiology (2014), 63, 923–930
072744G2014 The Authors Printed in Great Britain923
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N. Motamedi, L. Danelishvili and L. E. Bermudez
930 Journal of Medical Microbiology 63