# Host Peptidic Hormones Affecting Bacterial Biofilm Formation and Virulence

Olivier Lesouhaitier, Thomas Clamens, Thibaut Rosay, Florie Desriac, Mélissande Louis, Sophie Rodrigues, Andrei Gannesen, Vladimir k. Plakunov, Emeline Bouffartigues, Ali Tahrioui, et al.

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Olivier Lesouhaitier, Thomas Clamens, Thibaut Rosay, Florie Desriac, Mélissande Louis, et al. Host Peptidic Hormones Affecting Bacterial Biofilm Formation and Virulence. Journal of Innate Immunity, 2018, pp.1 - 15. 10.1159/000493926. hal-01930188

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# Host Peptidic Hormones Affecting Bacterial Biofilm Formation and Virulence

Olivier Lesouhaitierª Thomas Clamensª Thibaut Rosayª Florie Desriacª

Mélissande Louisª Sophie Rodriguesª Andrei Gannesenª⁻ᶜ

Vladimir K. Plakunovᵇ Emeline Bouffartiguesª Ali Tahriouiª Alexis Bazireᵈ

Alain Dufourᵈ Pierre Cornelisª Sylvie Chevalierª Marc G.J. Feuilloleyª

**Keywords**

Hormones · Peptides · Biofilm · Virulence · Mechanism of action · Bacterial sensor · *Pseudomonas aeruginosa* · Staphylococci

**Abstract**

Bacterial biofilms constitute a critical problem in hospitals, especially in resuscitation units or for immunocompromised patients, since bacteria embedded in their own matrix are not only protected against antibiotics but also develop resistant variant strains. In the last decade, an original approach to prevent biofilm formation has consisted of studying the antibacterial potential of host communication molecules. Thus, some of these compounds have been identified for their ability to modify the biofilm formation of both Gram-negative and Gram-positive bacteria. In addition to their effect on biofilm production, a detailed study of the mechanism of action of these human hormones on bacterial phys-

iology has allowed the identification of new bacterial pathways involved in biofilm formation. In this review, we focus on the impact of neuropeptidic hormones on bacteria, address some future therapeutic issues, and provide a new view of inter-kingdom communication.

© 2018 The Author(s)

Published by S. Karger AG, Basel

**Introduction**

Biofilms are defined as complex microbial communities of cells that are attached to a substratum (biotic or abiotic), an interface, or to each other and embedded into a matrix of self-produced extracellular polymeric compounds [1]. Biofilm formation endows bacteria with a high tolerance to antimicrobials and host immune defense mechanisms, thus enabling the pathogens to survive in hostile environments and disperse and colonize

<footnote>ª Laboratory of Microbiology Signals and Microenvironment, LMSM EA 4312, Normandy University, University of Rouen Normandy, Evreux, France; ᵇ Winogradsky Institute of Microbiology, Research Center of Biotechnology of RAS, Moscow, Russia; ᶜ Lomonosov Moscow State University, Moscow, Russia; ᵈ Laboratoire de Biotechnologie et Chimie Marines EA 3884, IUEM, Université de Bretagne-Sud (UBL), Lorient, France</footnote>
new niches. In addition, a small percentage of persister
cells developing within the biofilm is known to be highly
tolerant to antibiotics and has typically been involved in
causing the relapse of infections [2]. The medical conse-
quence for the infected host is the persistency of chronic
infections in organs in which the biofilm is established
[3]. While the molecular mechanisms that allow Gram-
negative bacteria to resist to antibiotics within biofilms
are well documented, this is not yet the case for Gram-
positive bacteria [4, 5]. Based on naturally occurring an-
timicrobial and host defense peptides, the design of small
synthetic peptides with broad-spectrum antibiofilm ac-
tivity has provided a glimmer of hope in the fight against
pan-antibiotic-resistant bacteria, the so-called superbugs
[6, 7], providing a really promising start for the develop-
ment of new antibacterial agents [8]. These peptides have
been tested either alone [9] or in association with antibi-
otics since they could act as biofilm-dispersive agents to
improve the action of antibiotics [10]. Some of these pep-
tides, whether endogenous or synthetic (i.e., LL-37 and
peptide 1018), do not require a specific binding to a bac-
terial target such as a sensor protein [9]. However, as for
antibiotics, some cationic antimicrobial peptides (AMP)
such as LL-37 or magainin II induce the emergence of
bacterial resistance often through the activation of 2-com-
ponent systems (TCS) [11–13].

In the search for new strategies or bacterial targets to
counteract bacterial infection and/or biofilm formation,
the potential as antimicrobials of eukaryotic communi-
cation factors released constantly by host cells (hor-
mones and cytokines) during a surgery (norepinephrine)
[14] or during infections (natriuretic peptides, substance
P [SP], or neuropeptide Y) [15–17] has emerged. This
concept originated in 1930, when it was suspected that
epinephrine could favor bacterial infections [18]. This
hypothesis was validated by the studies of Lyte and Ernst
[19], which showed that epinephrine and more generally
catecholamines enhance bacterial virulence. This work
opened a new research field named “microbial endocri-
nology” [20, 21] that refers to bacterial sensitivity to host
hormones. The discovery that the bacterial response to
epinephrine and norepinephrine was not only restricted
to *Escherichia coli* [22] suggested that this phenomenon
was widely encountered in host-bacteria relationships.
In addition, since in humans these catecholamines act
through specific eukaryotic receptors, it was tempting to
explore the possibility that the bacterial sensitivity to
these hormones was relayed via specific bacterial sensors.
In this context, a crucial step in the comprehension of
this mechanism was achieved by the studies of Sperandio

et al. [23] and Clarke et al. [24], since they identified
QseC as the bacterial sensor for catecholamines. In addi-
tion, this sensor further appeared to be widespread since
homologs of *E. coli* QseC have been referenced in 24 bac-
terial species [25]. Interestingly, it was observed that bac-
teria expressed several subtypes of catecholamine sen-
sors [26], as in human cells where numerous receptor
subtypes exist. In parallel to the characterization of cat-
echolamine effects on bacteria, several studies have fo-
cused on the impact of eukaryotic communication com-
pounds (i.e., neurotransmitters, cytokines, and hor-
mones) and a clear consensus emerged that numerous
eukaryotic molecules have a physiological effect on a
large variety of bacteria [27, 28] and that these activities
are quite often dependent on binding to a bacterial sen-
sor. For instance, it was shown that interferon-γ led to
the induction of the *Pseudomonas aeruginosa* lectin PA-I
via activation of the quorum-sensing network that is re-
quired for the production of virulence factors [29]. No-
ticeably, this effect was shown to be triggered by direct
binding of interferon-γ on OprF [29], which is a multi-
functional outer membrane porin [30] that is required
for *P. aeruginosa* virulence regulation [31] and involved
in regulation of biofilm formation [32]. It has also been
observed that dynorphin A, an endogenous opioid, mod-
ifies *P. aeruginosa* physiology [33]. It is interesting to
mention that opioid receptor agonists are able to mimic
the effect of dynorphin A on *P. aeruginosa*, especially by
enhancing bacterial adhesion properties [33], suggesting
that the effect of this human peptide is relayed by a bac-
terial sensor showing a pharmacological profile similar
to that of its eukaryotic counterpart [33]. In the same
vein, it was shown that morphine can trigger *P. aerugi-
nosa* virulence [34]. This seemingly exotic idea that bac-
teria would express sensor proteins presenting pharma-
cological activities similar to that of the eukaryote recep-
tors was already suggested for a human hormone by
Yamashita et al. [35]. In this case, somatostatin, which
binds to receptors localized on the eukaryotic cell mem-
brane that are coupled to adenylate or guanylate cyclases
[36], is able to modify *Helicobacter pylori* physiology by
enhancing the cAMP (cyclic adenosine monophosphate)
and/or cGMP (cyclic guanosine monophosphate) intra-
bacterial concentration [35]. This concept, in which hu-
man hormones may have an impact on bacteria through
bacterial cyclases, was also verified in *Pseudomonas* spe-
cies. Indeed, natriuretic peptides were shown to modify
the virulence of *P. aeruginosa* [37] and *P. fluorescens* [38]
after modulation of cAMP or cGMP intrabacterial con-
centration.
The data presented above show that the activation of bacterial sensors by host communication compounds results in the modulation of bacterial virulence, adhesion, and/or biofilm formation. Next to these physiological observations, identification of the putative bacterial sensors for these human hormones and the deciphering of the mechanisms of action of these hormones on bacteria could serve the scientific community to identify new bacterial targets to counteract bacterial virulence and biofilm formation. The originality of this review, which is focused on the neuropeptide hormone family, lies in the fact that it describes not only the impact of these compounds on biofilm formation and on virulence regulation but also the corresponding bacterial sensors and the mechanisms of action triggered by the binding of neuropeptide hormones on their target(s). This review will begin with the sensitivity of Gram-negative bacteria to host signal compounds and it will continue with the response of Gram-positive bacteria.

## Effects of Peptidic Hormones on Gram-Negative Bacteria

### Dynorphins and *P. aeruginosa*
Hormone Presentation

Dynorphins are involved among others in pain and stress responses after binding to the κ-opioid receptor family. Dynorphin A (1–17), dynorphin B (1–13) (Table 1), and α-neoendorphin are generated from the common precursor prodynorphin by proteolytic cleavage and share a highly conserved *N*-terminal sequence and charge distribution [39]. Since opioids have been shown to accumulate during inflammation [40], this highlights that bacteria can be exposed to dynorphins during infection.

### Impact of Dynorphins on *P. aeruginosa*

It has been shown that *P. aeruginosa* virulence increases after exposure to opioid agonists or dynorphins [33]. Indeed, the contact of *P. aeruginosa* with these compounds results in activation of the *Pseudomonas quinolone* signal (PQS) quorum-sensing system, pyocyanin (PCN) production, and consequently activation of bacterial virulence. More interestingly, exposure of *P. aeruginosa* to various concentrations of dynorphin A (100–400 µM) resulted in a dose-dependent effect on PCN production [33]. The biological relevance of this finding was further highlighted using a *Caenorhabditis elegans* nematode infection model, which showed enhanced virulence when *P. aeruginosa* cells were treated with dynorphins.

Since the 3 quorum-sensing-related signal compounds PQS, 4-hydroxy-2-heptylquinoline (HHQ), and 4-hydroxy-2-heptylquinoline-*N*-oxide (HQNO) were overproduced, the authors investigated the levels of PQS-regulated PA-I lectin mRNA [41, 42]. Since the authors observed that the synthetic κ-agonist U-50,488 mimicked the dynorphin effects on *P. aeruginosa* PCN and quorum-sensing quinolone signaling molecule production and on expression of the *pqSABCDE* operon, it has been speculated that, like U-50,488, dynorphins would increase the expression of lectin PA-I in *P. aeruginosa* by increasing the PQS level. This is underlined by the facts that: (1) the productions of lectin PA-I and PCN were described to be very similarly regulated, (2) PQS was previously shown to enhance the attachment of *P. aeruginosa* to stainless steel coupons [42], and (3) lectin PA-I is involved in attachment to surfaces and subsequent biofilm formation [43]. The effect of dynorphins was also assayed on preformed biofilm, but neither induction of rhamnolipids nor induction of the PQS system could be observed in this condition [44].

### Hormone Bacterial Targets and Mechanism of Action

The virulence and PQS production regulator MvfR was first suggested to be the bacterial sensor for dynorphins [33] (Fig. 1). This hypothesis is backed by the observation that MvfR is associated with the cytoplasmic membrane during the exponential phase of growth before being released into the cytoplasm by proteolytic degradation [45]. Recently, the effect of dynorphins on *P. aeruginosa* was suggested to be triggered by the TCS ParRS rather than by MvfR, independently of the quorum-sensing system [46]. Interestingly, ParRS TCS has been involved in AMP resistance [47] since ParRS was shown to be required for induction of the lipopolysaccharide (LPS) remodeling *arn* operon by the cationic cyclolipopeptide polymyxin B [47]. Two other mechanisms of multidrug resistance are attributed to the ParRS system: overproduction of the MexXY efflux pump and downregulation of the porin *oprD* gene, resulting in an active efflux of antibiotics and a decreased membrane permeability to imipenem, respectively [48] (Fig. 1). Since dynorphin was shown to bind directly the inner membrane (IM) sensor ParS leading to activation of the cytoplasmic regulator ParR, these data suggest that *P. aeruginosa* could perceive this host factor as a classical AMP and therefore adapt its physiological answer.

Although the dynorphin bacterial target and the associated mechanism of action of this peptide on *P. aeruginosa*
<table><caption>**Table 1.** List of the eukaryotic peptide hormones that have an effect on bacteria</caption><thead><tr><th>Peptides</th><th>Sequence and structure</th><th>UniProt/PubChem (CID)<sup>a</sup> accession numbers</th><th>Organs and cells that produce the peptide hormone</th><th>Bacteria encountered in the producing organ</th><th>Bacteria susceptible to the peptide (molecular target)</th><th>Key references</th></tr></thead><tbody><tr><td>Dynorphin A and dynorphin B</td><td>17 AA or 13 AA<br>YGGFLRRIRPKLKWDNQ<br>Dynorphin A structure</td><td>P01213/13133805/ 25081093</td><td>Cerebellum<sup>b</sup><br>Lymphoid node<sup>b</sup><br>Skeletal muscle<sup>b</sup><br>Gall bladder<sup>b</sup><br>Pancreas<sup>b</sup><br>Oral mucosa<sup>b</sup><br>Stomach<sup>b</sup><br>Intestine<sup>b</sup><br>Kidney<sup>b</sup><br>Testis<sup>b</sup><br>Placenta<sup>b</sup></td><td>Bacteria from the oral microbiota</td><td>P. aeruginosa (MvfR or ParS)</td><td>33, 39, 46</td></tr><tr><td>Somatostatin SST-14 and SST-28</td><td>14 AA (disulfide bridge 3–14] or 28 AA (disulfide bridge 17–28)<br>SANSNPAMAPRERKAGCKNFFWKTTFTSC SST-14 structure</td><td>P61278/16129681/ 16133849</td><td>Hypothalamus<sup>b</sup><br>Cerebral cortex<sup>b</sup><br>Adrenal cells<sup>b</sup><br>Pancreas<sup>b</sup><br>Stomach<sup>b</sup><br>Duodenum<sup>b</sup><br>Small intestine<sup>b</sup><br>Colon<sup>b</sup><br>Rectum<sup>b</sup></td><td>Bacteria from the intestinal microbiota</td><td>H. pylori (SSTR-2-like protein)</td><td>35, 51</td></tr><tr><td>ANP</td><td>28 AA (disulfide bridge 7–23)<br>SLRRSSCGGGRMDRIGAQSGLG CNSFRY</td><td>P01160 (human)/ 16166338 (rat)</td><td>Heart<sup>b</sup><br>Skin</td><td>Bacteria involved in endocarditis<br>Bacteria from the skin microbiota</td><td>S. epidermidis (molecular target not yet identified)<br>S. aureus (molecular target not yet identified)</td><td>110, 117–119</td></tr><tr><td>BNP</td><td>32 AA (disulfide bridge 10–26)<br>SPKMVQGSGCGFKRMDRISSSSGLGCKVLRRH</td><td>P16860/71308561</td><td>Heart<br>Skin</td><td>Bacteria involved in endocarditis</td><td>P. aeruginosa (molecular target not yet identified)</td><td>37, 117, 118</td></tr></tbody></table>
<table><caption>**Table 1 (continued)**</caption><thead><tr><th>Peptides</th><th>Sequence and structure</th><th>UniProt/PubChem (CID)<sup>a</sup> accession numbers</th><th>Organs and cells that produce the peptide hormone</th><th>Bacteria encountered in the producing organ</th><th>Bacteria susceptible to the peptide (molecular target)</th><th>Key references</th></tr></thead><tbody><tr><td>CNP</td><td>22 AA (disulfide bridge 10–26)<br>GLSKGCFGLKLDRIGSMSGLGC</td><td>P23582/16179407</td><td>Prostate (RNA)<br>Retina (RNA)<br>Testis (RNA)<br>Brain<br>Bone<br>Testis<br>Granulosa cells<br>Thyroid<br>Thymus<br>Skin<br>Endothelial cells</td><td>P. aeruginosa<br>S. aureus<br>Bacteria from the skin microbiota</td><td>P. aeruginosa (AmiC)<br>S. epidermidis (molecular target not yet identified)<br>S. aureus (molecular target not yet identified)</td><td>37, 68, 69, 77, 110, 117, 118, 120–123</td></tr><tr><td>Substance P</td><td>11 AA<br>RPKPQQFFGLM</td><td>P20366/36511</td><td>Cerebral cortex<sup>b</sup><br>Thyroid gland<sup>b</sup><br>Testis<sup>b</sup><br>Adrenal gland<sup>b</sup><br>Spleen<sup>b</sup><br>Bronchus<sup>b</sup><br>Nasopharynx<sup>b</sup><br>Pancreas<sup>b</sup><br>Salivary gland<sup>b</sup><br>Stomach<sup>b</sup><br>Intestine<sup>b</sup><br>Skin<sup>b</sup></td><td>Bacteria from the skin microbiota</td><td>B. cereus (EfTu)<br>S. epidermidis (EfTu)<br>S. aureus (EfTu)<br>P. fluorescens (molecular target not yet identified)</td><td>83, 88, 97, 99, 124</td></tr><tr><td>CGRP</td><td>37 AA<br>ACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF</td><td>P06881/56841902</td><td>Thyroid gland<sup>b</sup><br>Skin</td><td>Bacteria from the skin microbiota</td><td>S. epidermidis (DnaK)<br>S. aureus (EfTu)</td><td>83, 106, 109, 124</td></tr></tbody></table>

The amino sequence and the 2-D structure of each peptide, the UniProt accession number, and the PubChem accession number of the corresponding peptide, the organs and cells that produce the peptide (protein form or RNA), the family of bacteria encountered in the organs producing the corresponding peptide hormone, and the bacteria sensitive to the corresponding peptide and the bacterial molecular targets are shown. a Data retrieved from the National Center for Biotechnology Information PubChem Compound Database (https://pubchem.ncbi.nlm.nih.gov, accessed September 3, 2018) [125]. b Data retrieved from the Human Protein Atlas (https://www.proteinatlas.org/, accessed September 3, 2018) [126].
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Host peptidic hormones and Gram-negative bacteria. Model of the regulatory pathways involved in the effects of dynorphins on *P. aeruginosa* (a), somatostatin on *H. pylori* (b) and natriuretic peptides on *P. aeruginosa* (c). Arrows represent positive regulation and T-bar-finishing lines indicate negative regulation. OM, outer membrane; AC, adenylate cyclase; NAG, N-acetylglucosamine; NAM, N-acetylmuramic acid.</figcaption></figure>

*nosa* remain unclear, it appears that bacteria are able to sense the opioid dynorphin peptides, resulting in a complex bacterial response.

## Therapeutics Future

Opioid compounds are widely used in human health and affect the immune host defense [49]. A growing concern is related to the use of these compounds in humans as, like in animals, opioid use is associated with an increased risk of bacterial infections like pneumococcal dis-

eases [50]. A better understanding of how endogenous opioids as well as synthetic ones interact with bacterial cells may help in the identification of potential bacterial targets and the development of dual-therapy in high-risk patients. Knowing the structure-activity relationships between the bacterial sensor and opioids would lead to the development of compounds that might not be recognized by bacteria but that would keep their helpful effect on pain release in humans.
Somatostatin and H. pylori

Hormone Presentation

Somatostatins belong to a neuropeptide family that
has numerous functions, with effects on nearly every tis-
sue or organ of the human body, with a special mention
of the gastrointestinal tract. Somatostatins are produced
after proteolytic processing of a pre-pro-somatostatin
precursor. This process generates 2 cyclic peptides named
according to their length, i.e., somatostatin-14 (SST-14)
and somatostatin-28 (SST-28), that are characterized by
a 12-amino acid loop [51] (Table 1). Somatostatins exert
their biological effects after binding 1 of the 5 somatosta-
tin receptor (SSTR) subtypes (SSTR-1 to SSTR-5) ex-
pressed in humans [52, 53], which are coupled to an ad-
enylate cyclase through a G-protein.

Impact of Somatostatin on *H. pylori*

H. pylori is a bacterium that colonizes stomach and intestine mucosa where peptidic hormones such as somatostatin and gastrin are released. Gastrin peptides exist in 2 active forms (gastrin-17 and gastrin-34). A study conducted by Yamashita et al. [35] showed that the SST-14 somatostatin exerted a dose-dependent (1 µM to 10 pM) inhibition of H. pylori growth. In the same study, the authors concluded that the somatostatin effect on H. pylori was highly specific since gastrin peptides had no activity against H. pylori while somatostatin did not affect E. coli growth [35]. Nevertheless, this specificity should be confirmed since another study showed that gastrin could bind to a non-defined H. pylori sensor, increasing the bacterial growth rate [54]. Since somatostatin effects on human cells are relayed mainly by adenylate cyclases and occasionally by guanylate-cyclases modifying cAMP and cGMP intracellular concentrations, respectively, the impact of 2 cell-permeable analogs of cAMP and cGMP on H. pylori has been evaluated. Surprisingly, it has been observed that 8-bromo-cGMP fully mimicked the somatostatin inhibition of H. pylori growth while di-butyryl cAMP stimulated H. pylori growth, whereas cAMP and cGMP intrabacterial concentrations were both enhanced when H. pylori was exposed to somatostatin (10 pM; 48 h) [35]. These discrepancies suggested nonetheless that somatostatin acts on H. pylori through a specific sensor target.

Hormonal Bacterial Targets and Mechanism of Action

With the aim of characterizing the *H. pylori* soma-
tostatin sensor, binding studies using radiolabeled SST-
14 were performed. This enabled the identification of a

saturable site presenting a dissociation constant (K_D) of
0.31 nmol/L [35], confirming the presence of a specific
target on *H. pylori* for SST-14. In the same vein, using im-
munoglobulins directed against the SSTR subtypes, it has
been observed that IgG against SSTR-2 are able to block
somatostatin effects on *H. pylori* whereas IgG against
SSTR-1 have no impact [35]. Although these data remain
fragmentary since the bacterial target was not identified
at that time, they suggest the presence of a bacterial soma-
tostatin sensor presenting functional similarities with its
human counterpart SSTR-2 (Fig. 1).

Therapeutics Future

*H. pylori* biofilm establishment in the stomach is a
source of gastritis and peptic ulcers. In the search for nat-
ural compounds able to block *H. pylori* biofilm forma-
tion, curcumin has shown interesting potential [55].
However, it is important to keep in mind that curcumin
is able to reduce both somatostatin and gastrin release
[55]. Regarding the impact of these hormones on *H. py-
lori* described above, it is important to mention that all of
these parameters must be taken into account in the race
to discover natural compounds able to prevent stomach
disease.

Natriuretic Peptides and P. aeruginosa

Hormone Presentation

Natriuretic peptides constitute a family of hormones
first identified for their cardiovascular and osmoregula-
tion activities [56]. In addition to these well characterized
functions, numerous activities have been attributed to
these peptides [57]. The 3 main members of this family
are: atrial natriuretic peptide (ANP), brain natriuretic
peptide (BNP), the C-type natriuretic peptide (CNP),
which are mainly released in human blood by cardiomy-
ocytes (ANP and BNP) and endothelial cells (CNP). This
family of peptides is completed by urodilatin, uroguany-
lin, and guanylin, which are released by epithelial cells in
the intestine or in the kidney lumen, and dendroaspis na-
triuretic peptide, which is a component of snake venom
[58]. These peptides are synthesized as pre-pro-hormones
that are cleaved in the cytoplasm first as pro-hormones
and finally as the biologically active form by the action of
a membrane enzyme during the releasing process [57,
59–61]. The length of these peptides is between 22 and 38
amino acids [57] (Table 1), and their structure is charac-
terized by a disulfide bridge leading to a 17 amino-acid
loop conferring an omega form. This structure is encoun-
tered in some cationic AMP such as rhesus θ-defensin-1
from the macaque or tyrocidin from *Bacillus* [62–65].
These data added to the clinical observations showing that N-terminal-pro-BNP release is enhanced in the presence of *E. coli* or LPS in blood [15], and in vitro studies demonstrating that LPS added to cultured endothelial cells stimulated CNP release by these cells [66] have prompted some laboratories to study in more detail the impact of natriuretic peptides on the physiology of Gram-negative bacteria.

### Impact of Natriuretic Peptides on *P. aeruginosa*

The first work dedicated to the impact of natriuretic peptides on bacteria was performed by Krause et al. [67]. In that study, it was shown that, among the natriuretic peptides tested, human BNP (hBNP) possesses antimicrobial activity against both Gram-positive and Gram-negative bacteria and that hANP and urodelatin are active as well, but with a weaker activity, while hCNP does not show any direct activity against bacteria [67]. This work constituted a starting point to evaluate the antimicrobial potential of natriuretic peptides, even if the minimum inhibitory concentration measured for hBNP was around 5 µM against Gram-positive bacteria and around 30–35 µM against Gram-negative bacteria [67], which are concentrations still quite far above their physiological concentration in human blood [56].

In this context, study of the impact of lower concentrations of natriuretic peptides on bacterial physiology was required to evaluate their real potential. It was observed that, whereas hBNP and hCNP have no impact at 1 and 0.1 µM on *P. aeruginosa* growth [37, 68], bacterial exposure to these peptides at 1 µM stimulates the virulence of *P. aeruginosa* and *P. fluorescens* [37, 38]. In addition, both hBNP and hCNP at 0.1 µM strongly prevented *P. aeruginosa* biofilm formation [69]. The most active peptide was hCNP, which was able to impair biofilm formation in a dose-dependent manner, and which was still able at 10 nM to reduce by a half the biofilm formation by *P. aeruginosa* [68].

### Hormone Bacterial Targets and Mechanism of Action

In *P. aeruginosa*, production of virulence factors and biofilm establishment are inversely regulated at least partly by intracellular cyclic nucleotides [70]. The provirulent effect of CNP on *P. aeruginosa* was shown to result from the increase in the cAMP concentration [37], which activates the virulence factor regulator Vfr [71] (Fig. 1). In addition, in human cells, CNP was shown to bind both on the natriuretic peptide receptor (NPR) subtypes B (NPR-B) and C (NPR-C) [72], regulating cGMP

and cAMP intracellular concentrations, respectively [72, 73]. Altogether, these data suggested that a putative bacterial CNP sensor could be present in *P. aeruginosa*. This hypothesis was reinforced by a study showing that isatin, an antagonist of NPR-A and NPR-C receptors in rats, totally blocked the antibiofilm activity of CNP [69]. In this context, it was clearly suspected that the *P. aeruginosa* sensor for CNP could present some similarities with the eukaryotic NPR. An in silico search for protein structure similarities between the 3 types of human NPR (NPR-A, NPR-B, and NPR-C) and the *P. aeruginosa* whole proteome (www.pseudomonas.com) [74] was then performed. The best score was obtained for the acetamide sensor AmiC [75]. Comparisons of human NPR-C and *P. aeruginosa* AmiC in terms of amino acid distribution and 3-D model structures based on molecular docking approaches allowed the proposal of AmiC as a putative bacterial sensor for CNP [69]. This hypothesis was validated by microscale thermophoresis assays using purified AmiC, in which CNP was shown to directly bind AmiC with a $K_D$ of 2 µM [69]. It is interesting to note that this binding was highly specific since: (1) BNP displayed no affinity for AmiC, suggesting that the antibiofilm effects of BNP are mediated by another pathway in bacteria (Fig. 1), and (2) NPR-C but not NPR-A agonists can bind AmiC [69]. Altogether these data proved that a bacterial sensor for a hormone and its eukaryotic receptor counterpart could have similar pharmacological profiles.

The *amiC* gene belongs to the *ami* operon which contains 4 or 5 genes depending on the *P. aeruginosa* strain (www.pseudomonas.com) [74]. In the absence of acetamide, AmiC binds and sequesters the anti-terminator factor regulator AmiR [76]. When acetamide [76] or CNP [69] binds to AmiC, AmiR is released and the whole operon is overexpressed. It is tempting to propose that the aliphatic amidase AmiE, which is the final product of the *ami* operon, could be responsible for the antibiofilm effect of CNP on *P. aeruginosa*. This hypothesis was recently reinforced since it was demonstrated that overproduction of AmiE impaired *P. aeruginosa* biofilm formation [77] (Fig. 1). One outstanding issue remains: how and where does CNP bind AmiC in vivo? Up to now, AmiC was classified as being a cytoplasmic protein (www.pseudomonas.com) [74]. However, this protein possesses a periplasmic signature in its sequence, suggesting that AmiC could move to the IM. It has therefore been suggested that during CNP exposure *P. aeruginosa* would address a pool of AmiC close to or within the bacterial IM [69] (Fig. 1). In addition, it has been shown that the presence of a protein named PA4858, which presents an ami-
no acid sequence closely related to AmiC, is essential to
seeing the inhibitory effect of CNP on biofilm [69]. In this
context, AmiC could interact with CNP through the pu-
tative CNP periplasmic binding protein PA4858 [78]. It
is speculated that the protein PA4858 could finally facili-
tate binding between AmiC and CNP [69], probably
through a multiprotein complex including PA4859 to
PA4862 (Fig. 1).

Therapeutics Future

It was recently shown that the CNP concentration
used to affect *P. aeruginosa* physiology is important, since
CNP at 1 µM induced both specific effects through the
AmiC sensor and nonspecific effects likely through its in-
teraction with the bacterial membranes, triggering viru-
lence and/or antibiofilm activities [68] (Fig. 1). Converse-
ly, CNP concentrations of 0.1 µM or less inhibited biofilm
formation via binding to AmiC without an apparent viru-
lence increase [68]. These data suggest that CNP or CNP
derivatives could become interesting drugs for inhibition
of *P. aeruginosa* biofilm formation either alone or in as-
sociation with antibiotics, provided that a low hormone
concentration is used. Among CNP derivatives, the os-
teocrin peptide, which displayed an affinity for AmiC in
the nanomolar range, could be an interesting candidate
[69]. In any case, the enhancement of the bacterial AmiE
concentration induced after binding of CNP to AmiC
must also be taken into account since it was shown that
AmiE overproduction converts *P. aeruginosa* into a non-
virulent bacterium using an acute lung infection mouse
model [77]. Finally, we can speculate that the develop-
ment of such drugs could be facilitated by the fact that
natriuretic peptides are already used as therapeutic drugs.
Indeed, nesiritide (BNP) has been used since 2001 in pa-
tients with acute heart failure [79] and an agonist for na-
triuretic peptide receptors is actually in clinical trials
(phase IIa) for future use as a bronchodilatory drug [80]
and this could serve as a launch pad for delivery of natri-
uretic peptides in lung of patients suffering from *P. aeru-
ginosa* chronic infections.

Effects of Peptidic Hormones on Gram-Positive
Bacteria

The interest shown in the investigations on the effects
of neuropeptides on Gram-positive bacteria was driven
by the development of cosmetic sciences and the 1223UE
regulation which required, since 2009, a complete scien-
tific demonstration of all claims of the absence of adverse

effects (regulation 1223/2009). In this regard, the pres-
sure from consumers for preservative-free cosmetics or
cosmetic containing a minimum of preservatives was of
major concern since, in the absence of preservatives, any
substance could modulate the development, virulence,
and/or biofilm formation activity of skin-associated bac-
teria. The skin microbiota, which is the second most im-
portant one in the human body (i.e., 10¹² microorganisms
including more than 80% of bacteria) [81], was initially
regarded as a potential issue due to the presence of nu-
merous opportunistic pathogens. At this time, the whole
skin microbiota is considered as a potential new target of
cosmetic compounds. Cutaneous bacteria are continu-
ously exposed to sweat and a mean of 25% are localized
deep in the skin through hair follicles and sebaceous or
sweat glands [82]. Human skin represents the largest neu-
roendocrine organ of the human body [83], and cutane-
ous bacteria are continuously exposed to skin neuropep-
tides diffusing in sweat and though the tissue matrix [84,
85].

SP and Bacteria

Hormone Presentation

SP was the first skin neuropeptide whose activity to-
wards cutaneous bacteria was investigated. SP is an unde-
capeptide of the tachykinin family (Table 1), which is re-
leased in the skin by sensory C-type fibers [83]. It was the
first skin neuropeptide to be identified and it is also the
most abundant. Its role is not limited to neurotransmis-
sion and it is well known as a causative factor of neuro-
genic/antidromic inflammation, characterized by local
plasma extravasation and edema in the absence of any ex-
ogenous signal [86]. In eukaryotes, SP is recognized by
NK-1 receptors, although 2 other types of NK receptors
with a lower affinity also exist [87].

Impact of SP on Bacteria

The first study of the effect of SP on bacteria was per-
formed using a cutaneous strain of *Bacillus cereus* as a
model [88]. This species was not selected because of it
abundance in skin, although strains of *B. cereus* are part
of the commensal cutaneous flora [89], but because of its
potential virulence. Exposure of *Bacillus cereus* to SP was
associated with a rapid (5 min) and important (400 %)
increase in virulence that was associated with a significant
rise of biofilm thickness (+63% after 5 h) [88]. The sensi-
tivity of *B. cereus* to SP was considered high, since these
values were obtained at micromolar concentrations and
with a nanomolar threshold. The biofilm structure itself
was also affected by the exposure to the neuropeptide
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Host peptidic hormones and Gram-positive bacteria. Model of the regulatory pathways involved in the effects of SP on *B. cereus* (a), CGRP on *S. epidermidis* (b), and natriuretic peptides on staphylococci (c).</figcaption></figure>

with the formation of mushroom-like structures and a decrease in biofilm stability. The reversed sequence peptide of SP and neurokinin A, the natural ligand of human NK-2 receptors, did not show any activity, demonstrating the specificity of the action of SP peptides, suggesting the presence of a specific SP sensor in *B. cereus*.

Hormone Bacterial Targets and Mechanism of Action

The ligand of SP in *B. cereus* was identified by immunoprecipitation, Western blot and MALDI-TOF/TOF

analysis as the thermo unstable ribosomal elongation factor (EfTu) (Fig. 2). EfTu is produced in excess (4- to 14-fold) with regard to its stoichiometric association with ribosomes [90]. It is a member of a family of molecules identified as “moonlighting” proteins, i.e., proteins which possess alternative functions alongside their principal function. These different functions must be exercised by the same polypeptide chain and they must be realized into 2 different compartments of the cell. In this way, numerous enzymes or chaperones could be secreted outside of the cell or inserted into the membrane where they acquire
generally the role of environmental sensor [91–93]. EfTu
fits perfectly with this definition since this elongation fac-
tor is not only intracellular but it was also clearly found
on the cell membrane [94–96], suggesting that in addition
to its role in translation EfTu has a variety of other func-
tions. SP was found to display similar effects on biofilm
formation in the case of *Staphylococcus epidermidis* and
*S. aureus*, with an increase in thickness after exposure to
SP (1 µM) [97]. Like for *B. cereus*, EfTu was identified as
the SP sensor in *Staphylococcus* [97]. EfTu is a highly con-
served protein which is expressed in all bacteria as well as
in archaea and eukaryotes (in mitochondria) [98]. It was
thus tempting to hypothesize that SP is also capable of
acting on other bacterial species, including Gram-nega-
tive ones. So far, the only Gram-negative bacterium on
which the effect of SP has been investigated is *P. fluores-
cens* and it was observed that, as in Gram-positive species,
SP stimulated biofilm formation [99]. The target of SP in
*P. fluorescens* was not identified but it is interesting to
note that in *Pseudomonas*, including *P. aeruginosa*, EfTu
was also identified as a sensor for the neurotransmitter
γ-aminobutyric acid (GABA) [100, 101].

Therapeutics Future

Different complementary studies revealed that the ef-
fect of SP on biofilm formation can be inhibited by high-
ionic-force thermal water, probably through chelation,
and by cosmetic products able to block the access of SP to
the membrane [88]. The concentration of SP in skin and/
or sweat can be increased during inflammation, stress, or
even a nervous breakdown [84, 85] (these events are
known to be frequently associated with skin disorders).
Cutaneous bacteria, whose virulence and biofilm forma-
tion activities appear to be closely regulated by SP, are
likely to take part in skin alterations. This concept was at
the origin of the development of new cosmetic products,
as well as new drugs with dermatological clinical applica-
tions [102]. Indeed, it was shown that, in reconstructed
skin, SP-treated *S. epidermidis* promotes overexpression
of the keratinocytes integrin-α-5 and chemokine ligand
10, which are both typical markers of psoriasis, suggesting
that SP, in addition to its direct activity on skin cells [103],
should be somehow involved in the development of pso-
riasis through its action on skin bacteria [97]. In addition,
it has been observed that *S. aureus* biofilm formation and
its persistence are associated with numerous skin disor-
ders such as folliculitis decalvans [104], psoriasis, acne,
and rosacea [105], suggesting that the impact of SP on
bacteria biofilm should be evaluated in relation to these
skin disorders.

Calcitonin Gene-Related Peptide and Bacteria

Hormone Presentation

Calcitonin gene-related peptide (CGRP) is a 37 ami-
no-acid peptide (Table 1) cosecreted with SP by skin
nerve terminals and it presents functions quite similar to
those of SP in skin [83]. Considering its concomitant se-
cretion with SP, CGRP was logically the second neuro-
peptide whose potential action on cutaneous bacteria was
investigated.

Impact of CGRP on Bacteria

Until now, the effect of CGRP had only been investi-
gated in *S. aureus* and *S. epidermidis*, but the 2 Staphylo-
cocci behaved differently. Whereas *S. aureus* was not sen-
sitive to CGRP with no modification of bacterial viru-
lence regulation and biofilm formation even at the highest
concentration tested (1 µM), *S. epidermidis* showed an ex-
treme sensitivity to CGRP with a nanomolar threshold
[106]. In *S. epidermidis*, CGRP induced an increase in
virulence of the same range as that observed with SP,
which was followed by increased surface hydrophobicity
and a decrease in biofilm formation at least in dynamic
conditions [106] (Fig. 2).

Hormone Bacterial Targets and Mechanism of
Action

Unexpectedly, the effects of SP and CGRP on *S. epider-
midis* were not additive but antagonistic, suggesting that
both peptides were acting on a common and easily satu-
rable bacterial target. Surprisingly, this common step in
the mechanism of action of SP and CGRP was not the
recognition site, as CGRP was found to act through an-
other moonlighting protein, i.e., the chaperone DnaK
[106] (Fig. 2). However, the SP sensor EfTu and the CGRP
sensor DnaK share the fact that they do not have export
sequence signals and that, as shown in *E. coli*, these pro-
teins should be exported through the cytoplasmic mem-
brane by mechanosensitive channels (Msc) [107], pre-
sumably following transport by an MreB-dependent pro-
cess [108]. In line with the involvement of Msc channels,
blocking them using the specific inhibitor gadolinium
chloride inhibited both EfTu and DnaK export and mark-
edly inhibited the effects of SP and CGRP on *S. epidermi-
dis* [106]. The apparent absence of sensitivity of *S. aureus*
to CGRP remains a puzzling question. It should be noted
that DnaK is a much larger protein than EfTu (70 vs. 43
kDa) and that the pores formed by Msc channels in *S. epi-
dermidis* and *S. aureus* are not identical in terms of width.
Since the binding of SP and CGRP to their sensor should
occur in the periplasmic space (Fig. 2), DnaK cannot go
through Msc in *S. aureus* while EfTu can cross the pore because of it smaller size, explaining why the bacterium should be sensitive to SP (via EfTu) and not to CGRP as experimentally observed [109].

### Therapeutics Future

Until now the effect of CGRP on *S. epidermidis* has not been a matter of cosmetic or clinical applications, presumably as these observations were recent, but these data confirmed that skin neuropeptides should play a central role in the control of cutaneous bacterial behavior.

### Natriuretic Peptides and Gram-Positive Bacteria

We can consider that all known neuropeptides can be found in skin, although they should be only present in specific niches. This is the case of natriuretic peptides (see above), which are produced by endothelial cells of capillary vessels and can diffuse from blood at a close distance to capillaries. In this regard, natriuretic peptides should be particularly concentrated in a specific region of the hair follicle, i.e., the bulge, where capillary vessels are abundant. However, this region of the hair follicle is rich in sebum and it is colonized by bacteria such as *S. aureus* and *Cutibacterium (Propionibacterium) acnes* because of their lipophilic characteristic and tolerance to low oxygen local concentrations [81].

### Impact of Natriuretic Peptides on Gram-Positive Bacteria

It was recently demonstrated that the effects of natriuretic peptides on *S. aureus* were highly dependent on the growth temperature. At 37°C, the biofilm formation activity of *S. aureus* was inhibited by ANP and CNP, whereas that of *S. epidermidis* was increased [110]. The opposite effect was observed at 33°C (Fig. 2). These results should be correlated to the skin microenvironment since, while in depth the temperature stabilizes at 37°C, the surface temperature is generally about 33°C. Very recent data suggest that the biofilm formation activity of *C. acnes* should be submitted to even more complex temperature regulation, or probably medium- and oxygen-dependent regulation by natriuretic peptides [111], as was observed for *S. aureus*.

### Therapeutics Future

Natriuretic peptides, SP, and CGRP are known as key factors of neurogenic antidromic inflammation in skin. This process should be essential in atopic dermatitis [112], psoriasis [113], rosacea [114], and a large range of inflammation-related troubles [115]. The discovery of the

effects of SP, CGRP, and natriuretic peptides on Gram-positive bacteria led to a new vision of neurogenic inflammation which involves the microbial skin flora as a central relay in the inflammation process but also as a potential new target for the treatment of these diseases [116].

## Conclusion

Taken together, the original new field of research consisting of the study of the impact of eukaryotic communication signals on bacteria in general and biofilms especially suggests future applications mainly in cosmetology for Gram-positive bacteria and preferentially in the clinical field for Gram-negative bacteria. This review will encourage crossing of data obtained in these 2 application fields (i.e., cosmetic and clinical) in order to enlarge the spectrum of action of these peptides on bacteria. In addition, deciphering the mechanism of action of human hormones on bacteria will allow indirect discovery of new functions for bacterial proteins in term of virulence regulation or biofilm formation often far from the main functions for which they were primarily characterized, extending the concept of moonlighting proteins.

## Acknowledgement

T. Clamens and T. Rosay were recipients of a doctoral fellowship from the French Ministry of Research (MRE). This work was supported by grants from the Evreux Portes de Normandie, the Conseil Général de l'Eure, the European Union (FEDER), the Normandy region, and the French Association "Vaincre la Mucoviscidose."

## Disclosure Statement

The authors have no conflict of interests to declare.

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