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Editorial

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Do antimicrobial peptides and


antimicrobial-peptide resistance play
important roles during bacterial infection?
Gordon YC Cheung1 & Michael Otto*,1
1
Pathogen Molecular Genetics Section, Laboratory of Bacteriology, National Institute of Allergy & Infectious Diseases, US National
Institutes of Health, 50 South Drive, Bethesda, MD 20814, USA
*Author for correspondence: Tel.: + 301 761 6402; motto@niaid.nih.gov

“Nizet et al. reported that skin infection with group A streptococci was more severe in Cnlp
−/−
-knockout mice they had created, for the first time clearly indicating that the production of
AMPs matters in the control of bacterial infection”

First draft submitted: 30 April 2018; Accepted for publication: 15 May 2018; Published online:
16 August 2018

Keywords: antimicrobial peptide • antimicrobial resistance • Staphylococcus aureus

Bacteria are responsible for thousands of human deaths worldwide every year, a situation that to a large extent, is
due to antimicrobial resistance. However, humans are constantly confronted with potentially pathogenic bacteria
and only some bacterial infections progress to a state which requires medical intervention. Most infections can be
controlled in their initial phase by the human immune system. Serious infection only arises when the immune
system is overwhelmed by the sheer number of bacterial invaders or in the case of highly pathogenic strains
or efficient bacterial virulence mechanisms. Adaptive immunity, which is mostly based on specific antibodies
only comes into play after several days. In contrast, the innate immune system, which is activated by conserved
bacterial surface structures can react within hours to the invading bacteria. In the blood and tissues, mechanisms of
innate immunity that eliminate bacteria predominantly comprise phagocytes, such as neutrophils or macrophages.
Additional innate immune mechanisms exist on the epithelial and mucosal surfaces to control potentially harmful
colonizing bacteria like Staphylococcus aureus. These prevent bacteria from proliferating to an extent that would
enable them to overgrow and ultimately breach through the protective epithelial layers [1].
Antimicrobial peptides (AMPs) represent a major arm of innate immunity on epithelia, where they are secreted
mostly by keratinocytes [2,3]. In addition, AMPs contribute to the multifaceted killing mechanisms that work
together to eliminate bacteria within the phagosomes of neutrophils and other phagocytes. AMPs are a phylogenet-
ically early invention and are present in lower forms of life as well as humans. They can be structurally divergent and
form several different classes, including linear, bridged and more complicated, often posttranslationally modified
peptides. In humans, the most important AMPs belong to the defensin class, which can be further diversified
into alpha and beta defensins [4] and the cathelicidins [5], of which there is only one member found in humans,
called LL-37 [6]. Most AMPs are cationic and interact with the negatively charged bacterial surface. Therefore, the
term cationic AMPs is often used. There is a more limited number of negatively charged AMPs, such as the AMP
dermcidin, which have been isolated from the human skin [7].
Many AMPs have been investigated in extensive in vitro research, showing that they can kill bacteria using
divergent mechanisms. While many AMPs are pore formers, such as LL-37 or dermcidin [6,8], others like human
beta-defensin 3 additionally inhibit different essential parts of bacterial physiology, such as cell wall formation [9].
However, despite the profound mechanistic knowledge of AMP activities, it has remained unclear whether AMPs
play a key role in controlling bacterial infection. Considerable doubt about whether this is the case was based
mostly on the fact that during in vitro examination, the concentration of AMP that had to be used to kill bacteria,
significantly exceeds AMP concentrations present in phagosomes and, even more so, in the skin. As for the situation
on epithelia, it has been argued that local concentrations of AMPs during AMP–bacteria interaction may be higher

10.2217/fmb-2018-0138 
C 2018 Future Medicine Ltd Future Microbiol. (Epub ahead of print) ISSN 1746-0913
Editorial Cheung & Otto

than those found on average on the skin and most in vitro tests did not use buffer conditions representing an
epithelial environment. It was later shown that optimizing buffer conditions can lead to a lower concentration of
AMP being active in vitro [10], but the question whether AMPs really have a role in infection remained unanswered
for a long time.
The cathelicidin LL-37 (also called CAP-18 for cathelicidin antimicrobial peptide) is secreted by keratinocytes
and also present in phagosomes. It is cleaved from an 18-kD peptide precursor by kallikrein proteases to its
mature form with a length of 37 amino acids. LL-37 has been in the focus of research on the topic of AMPs in
bacterial infections and bacterial resistance mechanisms to AMPs. Mice have a homolog of LL-37 that is called
cathelicidin-related antimicrobial peptide (CAMP), which is encoded by the Cnlp gene. In a groundbreaking study,
Nizet et al. reported that skin infection with group A streptococci was more severe in Cnlp −/− -knockout mice they
had created, for the first time clearly indicating that the production of AMPs matters in the control of bacterial
infection [11]. Later, several other studies extended this observation to other bacteria and types of infection, all
using those knockout mice. For example, Chromek et al. showed that urinary tract infection by Escherichia coli [12]
and Danka & Hunstad showed that E. coli cystitis is dependent on the production of CAMP/LL-37 [13]. Huang
et al. demonstrated that Cnlp−/− mice exhibits increased susceptibility to Pseudomonas aeruginosa keratitis [14]. In
addition, Gupta et al. reported recently that pulmonary tuberculosis is affected by CAMP/LL-37 [15].
Antibiotic resistance has been shown to emerge sometimes within only a couple of years after the introduction
of an antibiotic into clinical use. In comparison, bacteria had millions of years to become resistant to AMPs
during their coevolution with humans and other AMP-producing organisms. Thus, not surprisingly, a plethora of
AMP resistance mechanisms are found in bacteria, and there are also structural adaptations in AMPs that can be
interpreted as an evolution to circumvent those resistance mechanisms [16]. For example, a very simple and general
bacterial AMP resistance mechanism is the production of secreted proteases and AMPs have evolved to counteract
proteolysis by forming proteolysis-resistant AMPs that are bridged or circular. However, there are many more AMP
resistance mechanisms in bacteria. Many involve the modification of the bacterial surface to make it less negatively
charged in order to minimize cationic AMP attraction. Gram-negative bacteria often modify lipopolysaccharide to
that effect, while Gram-positive bacteria modify teichoic acids with D-alanine, making them less anionic. Similarly,
the incorporation of lysinylated phospholipid reduces the negative charge of the bacterial membrane. Finally, efflux
pumps represent an efficient way for bacteria to dispose of many harmful substances; however, not many pumps have
been shown that accept AMPs as substrates. Some of the resistance/nodulation/cell-division family transporters,
which are present in many Gram-negative bacteria export AMPs such as LL-37 [17]. Recently, we described the first
Gram-positive export system with an AMP export function in S. aureus. This system was initially found to export
staphylococcal cytotoxins called phenol-soluble modulins and hence, was named Pmt (for PSM transporter) [18].
Until recently, direct evidence that AMP resistance mechanisms impact bacterial survival during infection had
not been provided. This is mostly due to the problematic situation, from an investigator’s point of view, that most
AMP resistance mechanisms also impact bacterial physiology in ways that are unrelated to AMPs, as they usually
comprise rather general adaptations, such as modifications of the cell surface or changes in secreted proteolytic
activity. In the 2001 Streptococcus study of Nizet et al. [11], a transposon mutant in a transcriptional regulator,
originating from a screen searching for LL-37-resistant isolates showed increased pathogenicity in mice. However,
it was not shown that this effect was AMP-dependent, nor was the function of that regulator – then named
cathelicidin resistance transcriptional regulator (CrgR) – determined. In fact, it can be assumed that a regulator
controls many physiological functions in addition to AMP resistance, making it difficult to use it as an example to
examine the role of bacterial AMP resistance in vitro or in vivo.
The situation of AMP-unrelated functions is less problematic for efflux pumps, which show higher degrees
of specificity. The Pmt system secretes AMPs and PSMs [19]; and thus, eliminating PSM production by genetic
engineering in S. aureus enabled us to directly address the key question of whether AMP resistance mechanisms
matter for bacterial infection. In that work, we could show that Pmt-positive S. aureus (devoid of PSMs) produced
significantly more pronounced skin infection in wild-type mice than an isogenic Pmt-negative strain, but that this
effect was not observed in Cnlp−/− (CAMP-deficient) mice [19]. This study thus showed for the first time that a
bacterial AMP resistance mechanism leads to more pronounced pathogenicity in vivo.
Altogether, investigations performed in the last several years have provided evidence substantiating the important
in vivo roles of both AMP production in the host to control bacterial infection and AMP resistance mechanisms
in the infecting bacteria. Going forward, this is an important information for current efforts to use AMPs as

10.2217/fmb-2018-0138 Future Microbiol. (Epub ahead of print) future science group


Antimicrobial peptides & antimicrobial-peptide resistance during bacterial infection Editorial

alternatives to antibiotics in an era of antibiotic resistance. Additionally, our recent research points to Pmt as a
cytotoxin exporter as well as AMP resistance mechanism as a promising antivirulence target [20].

Financial & competing interests disclosure


This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, US
National Institutes of Health. The authors have no other relevant affiliations or financial involvement with any organization or
entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from
those disclosed.
No writing assistance was utilized in the production of this manuscript.

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