Bacterial Peptides: Enormous Diversity
Peptides are defined as short chains of amino acids that are linked by peptide bonds.
In eukaryotes, peptides encompass an enormous range of structure and function, from
signaling hormones, to anti-pathogen molecules, to powerful toxins. In bacteria, ribosomally
produced peptides known as bacteriocins have been historically investigated for their
potential antimicrobial activities [1]. However, recent efforts in genomics and natural
product discovery have led to a tremendous “explosion” in the sheer number and diversity
of ribosomally produced peptides from the prokaryotic domain. It has been estimated
that all bacteria and archaea produce at least one, but more likely multiple, bacteriocin-like
peptides that have a wide range of functions including antimicrobial toxins, virulence
factors, and bacterial “hormones” that allow bacterial communities to organize multicellular
behaviors such as biofilm formation. This article provides an overview of ribosomally
produced bacterial peptides and their diverse roles in bacterial lifestyles, along
with future prospects and recent computational and bioinformatic approaches aimed
at decoding the overall “language” of these bacterially produced peptides.
Structure and Classification of Small Bacterial Peptides
Ribosomally produced bacterial peptides are a large class of compounds that encompass
an extraordinary amount of chemical, structural, and functional diversity (Figure
1) [2], [3]. These small peptides can range from unmodified linear forms to highly
modified, and sometimes circularized, structures. These modifications serve to confer
specific chemical properties that could not be obtained via peptide synthesis alone,
further increasing the number and complexity of these bacterial peptide families.
Furthermore, certain modifications are thought to serve as an important safety mechanism
to regulate the toxic activities of the bacterial peptide, thereby providing a level
of control and self-immunity [2], [4]. Some of the major chemical classifications
of ribosomally produced bacterial peptides include Lantibiotics such as Nisin, Linear
azo(line)-containing peptides such as Microcin B17, Lasso peptides such as the Escherichia
coli antibacterial peptide Microcin J25, and many others that continue to be discovered
at a rapid pace [5]. Approaches to systematically classify all known ribosomally produced
bacterial peptides have involved dividing groups based on: (1) particular prolific
producers such as lactic acid bacteria, (2) particular modifications of the bacterial
peptide, or (3) specific peptide activities. Indeed, given the sheer number and diversity
of these bacterially produced compounds, there is tremendous potential in the discovery
and development of these natural products as therapeutics. It has been noted that
with respect to bacteriocins, bacteria have, in essence, “already designed what clinicians
and pharmaceutical industries are once again struggling to obtain” [2].
10.1371/journal.ppat.1004221.g001
Figure 1
Functional diversity of ribosomally produced bacterial peptides.
Bacterial peptides produced by both gram-positive and gram-negative bacteria include
antimicrobial peptides such as Nisin and Microcin B17, known host virulence factors
such as the Streptolysin S-like cytolysins, and the peptide cytolysin from E. faecalis.
Bacterial peptides that structurally resemble bacteriocins are also utilized as signaling
molecules. Computational and genomic approaches, such as BAGEL, BACTIBASE, and Anti-SMASH,
can be combined with genomic data to catalog and discover new ribosomally produced
bacterial peptides. Combining computational approaches with experimental data can
guide the development of novel antimicrobials and artificially derived peptides with
specific functions and targets.
Bacterial Peptides as Antimicrobial Compounds
Many bacterial peptides have been known to exhibit potent antimicrobial activity against
competitor species of the producing microorganism. Nisin, whose activity was first
reported in 1928 [6], has been used widely in the food industry to prevent food-borne
pathogens [7]. The mechanism of Nisin's action is to bind to an intermediate in bacterial
cell wall synthesis, resulting in bacterial killing through pore formation [8]. Nisin
belongs to a larger group of bacteriocins known as lantibiotics; these compounds have
been highly studied, given their ability to specifically target clinically important
pathogens such as Streptococcus pneumoniae, Methicillin-resistant strains of Staphylococcus
aureus (MRSA), and Vancomycin-resistant enterococci (VRE) [9]. Microcin B17, a linear
peptide produced by particular strains of E. coli, is one of many bacteriocins that
are enzymatically modified by the addition of heterocyclic residues [10]. Microcin
B17 targets susceptible bacteria by inhibiting bacterial DNA gyrase [11]. In contrast
to Nisin, whose antimicrobial activity is most active against gram-positive bacteria,
Microcin B17 has been shown to be effective against a wide range of gram-negative
pathogens, such as E. coli, Salmonella, Shigella, and Yersinia species. A widely studied
example of an unmodified bacterial peptide is the enterococcal bacteriocin AS-48,
which has antimicrobial activity against gram-positive pathogens such as Listeria
monocytogenes
[12]. AS-48 bacteriocin belongs to a larger class of unmodified peptides that adopt
a particular native structure that is critical for their activity.
Bacterial Peptides as Virulence Factors
Recent discoveries have uncovered the fact that long-known potent bacterial toxins
such as Streptolysin S (SLS) are actually small, ribosomally produced peptides, whose
enzymatic modifications are highly similar to those of known bacteriocins, such as
Microcin B17. In addition to their well-described role in microbial warfare against
competing bacterial species, these peptide toxins are beginning to be recognized as
key contributors to initiating host disease. Streptolysin S has been identified as
a major contributing factor in successful translocation of Streptococcus pyogenes
across the epithelial barrier through a mechanism involving the disruption of intracellular
junctions via cleavage of occludin and E-cadherin [13]. The ability of peptide toxins
such as SLS to prevent phagocytic clearance can also be mediated through direct killing
of immune cells. A series of simple in vitro experiments exploring the effects of
SLS on mouse peritoneal macrophages in the early 1970s provided the first indication
that bacteriocin-like toxins can exhibit leukotoxic effects [14]. Like S. pyogenes,
Enterococcus faecalis also produces a peptide cytolysin (encoded by the cyIL gene
cluster) that is capable of lysing neutrophils and macrophages to avoid immune clearance
[15]. Interestingly, several microbial peptide toxins have also been shown to have
synergistic activity with other bacterial virulence factors, suggesting that, in fact,
these bacterial peptides may serve the dual role of causing direct damage to the host
while also increasing the overall virulence output. For example, Hung et al. utilized
a murine infection model to demonstrate that the peptide toxin SLS synergizes with
the unrelated streptococcal pyrogenic exotoxin B (SpeB) during infection to enhance
several features of pathogenesis, including inhibition of phagocytic clearance and
the induction of macrophage apoptosis [16]. In commensal bacteria such as Lactobacillus
plantarum, it has been shown that production of antimicrobial bacteriocins can modulate
the immune response of dendritic and peripheral blood mononuclear cells as well as
alter host cytokine profiles versus nonbacteriocin producing mutants [17].
Bacterial Peptides as Communication Signals
Many gram-positive bacteria use small peptides to communicate within a multicellular
community to regulate processes such as cellular density, biofilm formation, competence
for mating, and coordinated control of virulence [18]. Quorum sensing, the act of
bacterial communication via extracellular diffusible molecules, allows bacteria in
many cases to synchronize group behavior and facilitate coordinated events. In gram-negative
bacteria, N-acyl homoserine lactones have been extensively studied for their role
as communication molecules; however, in gram-positive bacteria, recent studies have
revealed that small peptides function as the predominant signaling molecule of choice.
One of the earliest discoveries of peptide pheromone signaling is the Agr system in
Staphylococcus aureus, which uses a small cyclic peptide, known as autoinducing peptide
(AIP), to communicate in a multicellular setting to control the expression of virulence
genes for a coordinated effect on host pathogenesis [19]. Additionally, three other
major groups of bacterial peptides have been studied for their roles in intercellular
communication, which are the Gly-Gly processed peptides, the RNPP systems peptides,
and the Rgg motif signaling peptides [20]. In some cases, reports are emerging that
these peptides control multiple behaviors in bacteria. In Streptococcus intermedius,
the quorum signal peptide known as competence stimulating peptide (CSP) was found
not only to be involved in regulating competence for natural transformation but also
to regulate and promote biofilm formation [21]. Multifunctional roles for bacteriocins
are becoming a wider and more accepted phenomenon. For example, reports have suggested
that Nisin can act both as an antimicrobial molecule and through an autocrine signaling
mechanism to potentiate Nisin production in a cell density–dependent manner [22].
Understanding the “Grammar” of Bacterial Peptides
Genome mining has been an important technological resource in the discovery of novel
natural products, such as bacteriocins. Bacteriocin-like peptides are highly attractive
candidates for genome mining, as these natural products are genetically encoded with
nearby genes encoding their corresponding modifying enzymes. Proximity to genes encoding
known modifying enzymes can aid in the identification of peptide biosynthesis gene
clusters [23]. In many cases, several metabolites have been identified from “cryptic”
or “orphan gene clusters” [24]. These cryptic gene clusters have demonstrated that
new, as-yet-uncharacterized enzymology is likely to be involved in the assembly of
the final natural product, likely leading to greater diversities of bacterial peptides
than have previously been appreciated.
Many web-based Bacteriocin gene mining and annotation tools have been developed to
aid in the identification, characterization, and classification of novel bacteriocins.
These include mining tools such as BAGEL (http://bagel.molgenrug.nl) and bacteriocin
repositories such as BACTIBASE (http://bactibase.pfba-lab-tun.org/main.php). Anti-SMASH
is a recently developed website that expands genome mining to not only bacteriocins
but a host of other genetically identifiable antibiotics and other secondary metabolites
[25]. Although these tools are rapidly expanding the repertoire of bacterially produced
peptides, one elusive goal has been to deduce the function of a given bacterial peptide
from the structural and genetic information alone. Recent computing approaches, however,
have begun to address the goal of using purely in silico approaches in order to predict
the specific activity of peptides and proteins. Loose et al. have postulated a “linguistic”
model for the design of antimicrobial peptides. The repeated usage of particular amino
acid sequences that are common to antimicrobial peptides led these investigators to
propose a certain grammar that governs the “language” of antimicrobial peptides [26].
In a recent study, Gupta et al. used a database of toxic and nontoxic peptides coupled
with machine learning and a quantitative matrix approach to predict whether a given
peptide will have toxic properties [27]. Their program, Toxipred, functions to essentially
predict the relative toxicity of a given peptide based on sequence alone. Although
still relatively new, programs such as these and others, combined with the rapid pace
of genomic discovery, will rapidly accelerate the pace of drug discovery in these
bacterial peptide families.
Conclusions
Ribosomally produced bacterial peptides have seen a recent surge in interest due to
new structural, biochemical, and microbiological tools, along with advances in genome
sequencing and bioinformatics. Computational algorithms and bacterial peptide databases
are rapidly growing as more of these compounds are discovered and deposited. Although
traditionally bacteriocins have been classified as having antimicrobial properties,
recent findings suggest that bacteria utilize bacteriocin-like peptides to perform
a myriad of roles, including intercellular communication, host colonization and manipulation,
as well as the exciting finding that these small peptides can have multiple functions
in a given microorganism. Computational-based approaches, coupled with experimental
data, will allow investigators to decipher the overall “language” of these ribosomally
produced bacterial peptides such that novel antimicrobials and artificially derived
peptides with specific functions and targets can be soon developed—provided that we
will be able to “speak its language.”