Resistance Mechanisms to Antimicrobial Peptides in Gram-Positive Bacteria

1Antimicrobial peptides, also known as host defense peptides (HDPs), are found in most life forms, being part of the innate immune system against pathogenic bacteria, fungi, parasites and viruses. Due to the alarming increase in antimicrobial resistance to the commonly used drugs around the world and the lack in discovery of new drugs and alternative treatments, there is a growing concern among the scientific community that in a near future, the current clinical approaches might not be able to deal effectively with microbial infections. Therefore, AMPs have been suggested as an alternative therapeutic strategy, in combination or as a replacement for traditional antibiotics.

2The development of antimicrobial resistance against AMPs is not as prevalent when compared to antibiotics, since AMPs targets are diverse and changes can interfere with the functionality of the cell, especially since the cell membrane is the main point of attack. However, bacteria can evolve quickly and grow resistant against AMPs in vitro. Another approach in the use of AMPs is combination with traditional antibiotics, since both have shown to synergize, reducing microbial resistance. A few AMPs have been translated into the clinic; polymyxins B, bacitracin, gramicidin S, daptomycin and vancomycin have been used for treatment of several types of bacteria. However, a number of questions are yet to be answered, such as the toxicity and stability in vivo of many peptides, as thoroughly reviewed. The contact with human cells, such as erythrocytes, was shown to inhibit the activity of AMPs. Furthermore, physiological conditions of the host can interfere with the effectiveness of these molecules, along with the peptide’s pharmacokinetics. Though these are significant challenges, AMPs remain an interesting strategy and still expanding field, as many studies have tried molecular engineering as an approach to solve the concerns cited above. One such example is the production of synthetic D-enantiomeric peptides to avoid proteolytic degradation. So far, over three thousand different peptides have been identified, distributed among six different kingdoms (animalia, archaea, bacteria, fungi, plantae, protist), according to the Antimicrobial Peptide Database (APD) (aps.unmc.edu/AP/). In humans, over 130 peptides have been described, and while the vast majority has been tested as potential antimicrobial drugs, AMPs have a larger impact than just direct antimicrobial effects, actively engaging with the host immune system, modulating its activity, promoting chemotaxis and cell recruitment, meddling with the inflammatory and wound healing pathways, among many different functions. AMPs were also shown to have an anticarcinogenic effect, as extensively reviewed.

3An important group of antimicrobial peptides is the cathelicidins. The human representant of this group is LL-37, a cationic, amphipathic peptide, composed by 37 amino acid residues. Its precursor, hCAP18, was first isolated in neutrophils but can also be found in other cells, such as keratinocytes and mast cells. After its cleavage by neutrophil proteases, the peptide acquires its functional form. LL-37 effects have been extensively investigated, and include direct antimicrobial activity and immune modulation. Cathelicidins are also found in many vertebrates, including farm animals, birds, reptiles and fish. Indolicidin, a 13 amino acid peptide expressed in bovine neutrophils, has antimicrobial activity against Gram-positive and Gram-negative bacteria.

4Another class of cationic and amphipathic antimicrobial peptides is the defensins, which can be divided in three main groups: α-defensins, β-defensins and θ-defensins. In humans, only α- and β-defensins can be found, while θ-defensins are present exclusively in Old World primates. Among human α-defensins, there are six peptides expressed: Human Neutrophil Peptide (HNP) 1 through 4 and Human Defensins (HD) 5 and 6. α-defensins can be found in many different tissues such as the gastrointestinal and respiratory epithelia, female reproductive tract and blood cells. These peptides display direct antimicrobial activities and immunomodulatory effects, including chemotaxis. β-defensins are expressed mainly in epithelial cells but also in monocytes, macrophages and dendritic cells and have an important role regulating the host microbiome.

5Human Lactoferrin (hLF) is an 80 kDa bilobal glycoprotein, present in bodily fluids and neutrophils, which acts in the transport of metal ions, especially ferric iron (Fe3+). hLF displays a bacteriostatic effect through iron chelation, decreasing the extracellular concentration of this ion available to the microorganism. Furthermore, the iron-free molecule, Apolactoferrin (ApoLF), is able to interact with microbial cellular membranes, undergoing subsequent proteolysis which results in release of smaller and more potent cationic peptides, especially those found in the N-terminal lobe: Lactoferricin (LFcin), Lactoferrampin (LFampin) and LF1-11.

6Human lysozyme, also named N-acetylmuramide glycanhydrolase, is often cited as the first antimicrobial protein discovered and is extensively used in industry. Lysozyme is a 14 kDa enzyme that binds to cell wall peptidoglycans, cleaving the links between different sugars, thus inducing cell rupture. Similarly, to lactoferrin, peptides derived from the cleavage of lysozyme exhibit antimicrobial activity against Gram-positive and Gram-negative bacteria.

7With an array of antimicrobial peptides being produced by different human cells, bacteria have developed a number of strategies to prevent AMP binding, to avoid their lytic effects or to degrade the peptides, in order to thrive in the human host. In the next sections, the different mechanisms employed by Gram-positive bacteria to circumvent AMP action will be explored. Table 1 and Figure 1 summarize the resistance mechanisms employed by these bacteria.

8Gram-positive bacilli include some pathogenic, anaerobic spore-forming species, such as Clostridium spp., Listeria monocytogenes, Bacillus anthracis, and Bacillus cereus.

9The genus Clostridium is composed of about 15 pathogenic species, of which the most common are Clostridium difficile, Clostridium perfringens, Clostridium tetani, and Clostridium botulinum. Although these species are similar, the pathologies caused by them are diverse. C. tetani produces the tetanus neurotoxin (TeNT) that causes neurological disease (tetanus), characterized by muscle spasms and spastic paralysis of the limb muscles. C. botulinum produces the potent botulinum neurotoxin that causes a serious and fatal neuro-paralytic disease in humans and animals (botulism). C. difficile is the main causative agent of nosocomial diarrhea and gastroenteritis, which can lead to the development of asymptomatic or symptomatic diseases. Infection by C. difficile (ICD) has been increasingly reported in the United States. C. perfringens can also cause acute diarrhea, with an estimated death toll of 200,000 each year in Nigeria according to The World Health Organization (WHO).

10Listeria monocytogenes is a foodborne pathogen that causes gastroenteritis in immunocompromised individuals, children, pregnant women and the elderly. L. monocytogenes outbreaks in South Africa have reported around 1000 confirmed cases and 200 deaths in 2017–2018; in the United States, the bacterium was the causative agent in 147 confirmed cases and 33 deaths, making it the third most expensive foodborne pathogen in 2010, after C. botulinum.

11B. anthracis is the causative agent of anthrax and can manifest in four ways, namely: cutaneous, inhalation, gastrointestinal or injectable. B. cereus causes foodborne diseases, such as gastrointestinal, diarrhea and emesis. In 2016, the European Union (EU) reported about 413 food-borne outbreaks caused by Bacillus toxins that affected 6657 people, ranking it the second most common cause of food-borne outbreaks in that year.

12Since one of the most important mechanisms of AMP-based killing is the interaction with the negatively charged membrane, changing the membrane composition is a strategy used by many bacteria to survive AMPs action. Among those changes, the insertion of D-alanine in the lipoteichoic acids, a process named D-alanylation, is used to reduce the negative membrane charge, thus inhibiting interaction with AMPs. This resistance mechanism is regulated by the dlt operon, and it has been described in several Bacillus species, such as B. cereus, B. anthracis, B. thuringiensis and B. subtilis, L. monocytogenes, C. difficile, C. butyricum, Lactobacillus plantarum, and Lactobacillus reuteri.

13Another mechanism of envelope modification is called lysinylation of the membrane; it consists of addition of L-lysine to the phosphatidylglycerol. A protein called MprF is essential for membrane lysinylation. In B. anthracis, a strain deficient in MprF was more susceptible to LL-37 and HNP-1 when compared with the wild type strain. In L. monocytogenes, MprF was shown to be essential in protection against gallidermin, HNP-1 and HNP-2.

14Bacteria can modify cell wall components, such as the peptidoglycan. O-acetylation of the peptidoglycan is able to reduce killing by lysozyme in L. monocytogenes and B. anthracis. Modifications on cell wall constituents also include the N-deacetylation of the peptidoglycan and the glycosylation of the wall teichoic acids, which in L. monocytogenes and B. cereus is crucial to protect against lysozyme, LL-37 and CRAMP, a cathelicidins found in mice.

15In B. subtilis, deacetylation of the N-acetylmuramic acid by the protein PdaC confers resistance against lysozyme attack. Another protein, PdgA described in L. monocytogenes is responsible for a similar resistance mechanism inducing N-acetylation of the peptidoglycan; a study by showed that a L. monocytogenes strain lacking PdgA was more susceptible to lysozyme and mutanolysin.

16Changes in lipid composition are able to interfere with AMPs action, as shown for L. monocytogenes, where different proportions of lipids are found in bacteriocin resistant strains. In Listeria innocua, changes in the proton motive force, via FoF1 ATPase, which altered the membrane potential were related with resistance to nisin.

17A B. subtilis mutant resistant to daptomycin presents an irregular and more cationic membrane than the wild type, due to mutations in pgsA gene. The PgsA protein is responsible for the addition of phosphatidylglycerol to the membrane; in that sense, the diminished phosphatidylglycerol synthase function in the mutant strain was responsible for the increased resistance to daptomycin.

18A strategy employed by many bacterial species to evade antimicrobial host defense is by expelling the molecules using efflux pumps or ABC transporters. The same mechanism has been implicated in AMP expulsion.

19Subtilin is an antibiotic produced by B. subtilis; to avoid self-destruction, the bacterium possess an ABC transporter called SpaIFEG, this transporter ejects the subtilin to the extracellular environment. B. licheniformis is capable of producing bacitracin, an antibacterial peptide also produced by other Bacilli; similarly, to B. subtilis, the bacterium is immune to the antimicrobial due to the action of the BcrABC transporter, which ejects the AMP before it affects the producer cell.

20In C. difficile, the cpr operon is responsible for the extracellular transport of peptides. A similar ABC transporter, AnrAB, is also found in L. monocytogenes, able to export AMPs and antimicrobials, hence hindering their efficiency. B. subtilis also has similar detoxification systems, BceAB-RS, PsdRS-AB (also named Yvc-PQ-RS) and YxdJK-LM. These transporters are important for resistance and cell wall stress signaling against AMPs and antimicrobial drugs, such as bacitracin and lantibiotics. The YtsCD ABC transporter, independently or in association with YwoA, is responsible for bacitracin resistance in B. subtilis.

21Cell wall signaling can trigger the expression of many resistance-related genes such as sigma (σ) factors and global regulators in bacteria. In L. monocytogenes, sigma factors σB and σL and regulators such as VirR and LiaR regulate the expression of many virulence genes, such as the dlt operon, MprF – a protein responsible for adding L-lysine to membrane phospholipids–and ABC transporters, contributing to antimicrobial resistance. Lia-related regulators are also present in B. subtilis; in the presence of peptides that target the cell envelope, the stress sensor is activated and induces the expression of resistance genes such as LiaRS and other membrane modification genes.

22Clostridium difficile gene expression is also altered in the presence of AMPs. LL-37 induces overexpression of genes related to crucial functions; including those involved with cell wall and envelope homeostasis, ABC transporters and lysine metabolism; similarly, bacitracin and lysozyme can alter the expression of extracellular σ factors.

23The mannose phosphotransferase (Man-PTS) pathway is an important resistance mechanism against bacteriocins. In L. monocytogenes, the activation of the Man-PTS pathway led to changes in metabolism, alteration of the membrane charge and addition of alanine in teichoic acids in strains resistant to class IIa bacteriocin. Although the Man-PTS pathway is a target for bacteriocins, in Lactococcus lactis and Lactococcus garvieae, it was shown to participate in resistance mechanisms, specifically in combination with LciA. This pathway is also involved in bacteriocin resistance in L. plantarum, Leuconostoc mesenteroides, Lactobacillus salivarius, and Lactobacillus acidophilus, in combination with PedB, a protein that provides protection against the bacteriocin pediocin PA-1, in a complex, being able to avoid cell lysis by this AMP. Similarly, in Listeria innocua, overexpression of pedB generated a more resistant phenotype. In L. innocua, Man-PTS is regulated by a transcriptional activator (lin0142); inactivation of lin0142 is related to resistance to pediocin.

24The B. subtilis sigma factor V (sigV) is activated in presence of Lysozyme, regulating important resistance genes such as oatA, dltABCD, and pbpX, promoting protection by virtue of membrane alterations. The alternative sigma factor 54 (rpoN) is relevant in mesentericin Y105 resistance in Listeria monocytogenes; strains lacking the monocistronic unit of rpoN showed a higher susceptibility to this AMP - a phenotype reverted after complementation - indicating that the resistance genes are under regulation of rpoN.

25The genus Staphylococcus is responsible for various infections in humans like impetigo, scalded skin syndrome, toxic shock syndrome, pneumonia, endocarditis, urinary tract infections, and many others. The most clinically relevant members of this genus are Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus. They are grape-shaped, catalase producing Gram-positive spherical cocci. S. aureus are classified as coagulase positive, while S. epidermidis and S. saprophyticus do not show coagulase activity. Another trait shared by many staphylococci species is the presence of a carotenoid pigment called staphyloxanthin, which gives the colonies a golden color and has an inhibitory role against microbicide molecules and reactive oxygen species (ROS). Among their antigenic structures are the protein A, which binds to Fc region of immunoglobulin G (IgG) and prevents complement activation; the teichoic acids, which modulate mucosal adhesion and induce toxic shock through release of interleukin 1 (IL-1) and tumor necrosis factor (TNF); and polysaccharide capsule, with 11 different serotypes. Defense against AMPs in Staphylococcus spp.

26Staphylococcus aureus is able to prevent AMP-mediated killing through modifications of the phosphatidylglycerol in the bacterial membrane by the multiple peptide resistance factor protein (MprF). The protein promotes the reaction of phosphatidylglycerol with lysin, generating lysylphosphatidylglycerol (Lys-PG), which is then translocated to the outer leaflet of the membrane. This results in a shift in membrane charge, and a subsequent repulsion of cationic AMPs.

27The enhanced synthesis of the cationic phospholipid Lys-PG promotes changes in membrane fluidity also associated with increased resistance against different classes of AMPs in staphylococci. A study investigating the development of bacterial resistance to antimicrobial peptides demonstrated that exposure of S. aureus cultures to sub-lethal concentrations of magainin 2 and gramicidin D over several passages in vitro promoted resistance to these AMPs. The bacterial membrane adaptations induced by AMP exposure included an increase in net charge and altered membrane rigidity.

28Similarly, resistance to platelet microbicidal proteins (PMPs) in S. aureus has been linked with adaptations affecting membrane fluidity. A study investigating the mechanisms underlying S. aureus susceptibility to thrombin-induced PMP (tPMP-1) demonstrated that mutant strains with increased resistance to this AMP (either naturally occurring or artificially generated) displayed a high content of unsaturated lipids with longer chains, which led to an enhanced membrane fluidity. Interestingly, tPMP resistance in S. aureus correlated with an increased virulence in both human and experimental endocarditis, highlighting the importance of this AMP in controlling S. aureus infection.

29Resistance to cationic AMPs has also been associated with modifications in the cell wall teichoic acid by esterification with D-alanine, through the dlt operon, which reduces the net negative charge of the molecule. In S. aureus and S. xylosus, deletions of parts of the dlt operon induced a higher sensitivity to a variety of AMPs when compared to wild type strains. Interestingly, the increased susceptibility of the mutant strains were limited to cationic peptides, suggesting that electrostatic repulsion may be involved in resistance to cAMPs in S. aureus.

30The presence of carotenoid pigments is another described mechanism of AMP resistance in staphylococci. These molecules are depicted as virulence factors, for their protective role against oxidative host defense mechanisms. Evidence suggests staphylococcal carotenoids can also provide protection against different antimicrobial peptides, through their effect on cell membrane stability. In that work, a mutant strain with a defect in staphyloxanthin synthesis was compared with its supplemented counterpart in terms of susceptibility to a range of antimicrobial agents, including human HNP-1, PMPs, and polymyxin B. The supplemented strain showed a reduced susceptibility to the AMPs, which in this case was linked to a higher rigidity in the cell membrane. This apparent contrast with previous work showing a positive correlation between membrane fluidity and AMP resistance evidences the intricate balance driving peptide-cell membrane interactions. In that sense, extremes in rigidity or fluidity may hinder AMP insertion in the bacterial membrane.

31Another mechanism of S. aureus evasion from AMPs is trapping them by surface or secreted proteins and polysaccharides. S. aureus secrets a plasminogen activating protein, staphylokinase (SK), which converts it into plasmin. High concentrations of plasmin on the bacterial surface promote fibrinolysis, favoring tissue invasion and dissemination. It has been shown that SK can bind to and inactivate mCRAMPs (cathelicidin murine antimicrobial peptides) and α-defensins secreted by neutrophils, including HNP 1-3 reducing the activity of AMPs in 80%. In an in vivo trial with mice, S. aureus strains expressing SK were more resistant to α-defensin. Similarly, addition of purified SK was able to increase survival of strains that did not produce this protein in presence of α-defensin, in vitro.

32Staphylococcus epidermidis synthesizes the exopolysaccharide intercellular adhesin (PIA), a positively charged polymer of the extracellular matrix in biofilms, which can promote hemagglutination. Studies using mutant strains lacking this polysaccharide have shown a role for PIA in resistance to LL-37 and β-defensin (HBD-3). The mechanism responsible for PIA-mediated protection against AMPs seems to involve electrostatic repulsion, since the lytic activities of these antimicrobial peptides are dependent on the salt concentrations. Besides the protective effect against AMPs produced in the skin, PIA can also limit destruction of Staphylococcus by neutrophils, by forming a mechanical barrier that prevents bacterial uptake by phagocytes.

33Additionally, S. aureus is able to sequester iron from the heme site of hemoglobin through the Iron-regulated surface determinant (Isd), which is then released into the cytoplasm for metabolization. This ability is responsible for the bacterial resistance to the bacteriostatic effects of lactoferrin and other iron-binding peptides. Furthermore, resistance to the bactericidal action of lactoferricin can be induced in S. aureus by growing the bacterium in increasing peptide concentrations–which also promoted cross-resistance to other antimicrobials, like indolicidin and penicillin G.

34Antimicrobial peptides are relatively resistant to bacterial surface or secreted proteases, yet some proteases can cleave a broad spectrum of AMPs; one such example is aureolysin, which inactivates LL-37 by cleaving peptide bonds in its C-terminus, between residues Arg19-Ile20, Arg23-Ile24, and Leu31-Val32. Thus, Aureolysin expression allows a higher survival in environments with high concentrations of LL-37 such as the phagolysosomes in macrophages and neutrophils.

35Staphylococcus aureus displays a phenotype known as small colony variant (SCV), which has been associated with persistent skin infections. This phenotypic change allows S. aureus strains to evade innate immune responses, one of those being AMPs, since in SCVs, a higher MIC was observed.

36Analysis of four different AMPs found on the skin (beta-defensin – hBD-2 and -3, RNase 7, and LL-37) showed that SCV were more resistant to AMPs when compared with the wild type strains. Similarly, a mutant strain with a hemin biosynthesis gene deletion, hemB, displaying a SCV phenotype, was less susceptible to three of the four AMPs tested, when compared with its complemented mutant exhibiting normal phenotype. These results suggest that phase variation may be a mechanism of bacterial resistance to AMPs. This effect could be attributed to differences in membrane charge in the SCV strains, as suggested by, however, in that work, SCVs showed an increased resistance to only a fraction of the AMPs tested.

37Staphylococcus aureus expresses an AMP recognition system named Antimicrobial Peptide Sensor; this system comprises a sensor histidine kinase (ApsS), a DNA-binding response regulator (ApsR) and ApsX, responsible for interacting with the AMP. The ApsRSX regulators are responsible for the regulation of important genes related to AMP resistance, such as mprF, vraFG and the dlt operon. Aps from S. epidermidis has shown the ability to interact with a broad variety of AMPs; in contrast to S. aureus in which Aps are active over a more limited spectrum of peptides. The ApsS is a transmembrane protein with an extracellular group sensitive to AMPs, composed by nine amino acids with a negative charge, which binds to AMPs and rapidly inactivates them.

38The GraRS regulators induce the expression of mprF and dltABCD, when activated together with vraFG, as a response to AMPs and glycopeptides, whereas mutant strains negative for graRS or vraFG were more susceptible to the peptides as the surface alterations generated as protection mechanisms were reduced.

39The agr global transcriptional regulator induced a super expression of dltD, a member of the dlt operon. Another regulator is the LytSR, a transmembrane electrical potential sensor. AMP cell wall damage is partially due to changes in membrane polarization; therefore, deletion of LytSR increased susceptibility to HNP-1 and tPMPs, Interestingly, no conformational changes were found in mutant cells membrane, indicating an alternative resistance pathway.

40The VraTSR is a bacterial sensor which responds to stress; it is involved in S. aureus resistance do methicillin (MRSA) and other antimicrobials that target the cell wall. Exposure to AMPs activates operons VraSR e VraDE, leading to a change in the transcriptional profile with the repression of virulence and metabolism genes, and an induction of genes that regulate envelope homeostasis.

41Enterococci are a group of Gram-positive cocci comprising more than 30 species, of which E. faecalis and Enterococcus faecium are the most clinically relevant (Fiore et al., 2019). They can be found in several environments such as water, soil and food, and are able to colonize the gastrointestinal tract of different animals. Enterococci are a leading cause of nosocomial infections – including endocarditis, urinary tract infections and bacteremia–being responsible for 14% of hospital infections in the United States. The problem is aggravated by the increased intrinsic resistance and tolerance exhibited by these bacteria against several commercial antimicrobial agents, including β–lactams such as cephalosporins, and vancomycin. In addition, enterococci rapidly acquire resistance to many classes of antibiotics upon treatment, thus posing a great public health threat.

42Similarly, to many other species previously cited, enterococci reshape their cell envelope composition in response to AMPs, with lysinated phosphatidylglycerol, addition of D-alanine to teichoic acids via dlt or MprF, also, the N-acetylglucosamine deacetylase PdgA (EF1843) contributes to lysozyme resistance in E. faecalis, by promoting peptidoglycan deacetylation.

43Many Enterococci species are able to perform O-acetylation of the cell wall peptidoglycan a mechanism related with resistance to lysozyme.

44Proteases and inhibitors are found in E. faecalis, either degrading or binding to the peptide, preventing their lytic effects. Among the proteases, GelE and SerE, a gelatinase and a serine protease, respectively, are able to degrade LL-37, HYL-20 – an α-helical amphipathic analog of a natural AMP present in bees – and GL13K, a peptide found in human saliva. In addition, extracellular dermatan sulfate – a product released from proteoglycans after the activity of extracellular proteinases – was able to inhibit the activity of HNP-1 on E. faecalis, representing an important virulence mechanism for this bacterium.

45The Bcr transporter family is related to bacitracin resistance and is found in many enterococci species. In E. faecalis, BcrABD is an ABC transporter expressed in the presence of bacitracin. It is regulated by BcrR, which is responsible for the extracellular pumping of the polypeptide. However, the BcrAB is not the only mechanism of bacitracin resistance in E. faecalis; other two-component regulatory systems and ABC transporters were also described. In S. aureus, LtnIFE is responsible for protection against lacticin. E. faecium possess homologs with similar function.

46In E. faecalis and E. faecium, the Man-PTS pathway is also related to resistance against bacteriocins, however, there are several implications in metabolic pathways which could hinder the host colonization. Undecaprenyl pyrophosphate phosphatase (UppP) is also related to bacitracin resistance in E. faecium by reducing the amount of substrate for bacitracin-mediated cell death. Another regulator crucial for successful host colonization is the sigma factor SigV, which is involved in resistance to lysozyme, but not to nisin. rpoN is responsible for encoding the sigma factor 54 in E. faecalis, an important factor for bacteriocin resistance. Interestingly, sensibility to other AMPs did not change in absence of this sigma factor.

47Both E. faecalis and E. faecium share the LiaFSR stress-induced regulatory pathway. LiaFS is the homolog of VraTS from S. aureus. Strains lacking liaR showed higher sensitivity against daptomycin, and LL-37, HBD-3, nisin, gallidermin–a type A lantibiotic, the synthetic antimicrobial peptide RP-1, mersacidin–a type B lantibiotic and friulimicin, a cationic lipopeptide in E. faecalis. The deletion of liaF, along with gdpD, promoted a similar increase in resistance against daptomycin. The liaFSR and related genes, such as liaX, a sensor that inhibits LiaFSR, are directly related to the cell envelope alterations in response to antimicrobials.

48Group A Streptococci (GAS) includes bacterial species such as Streptococcus pyogenes and Streptococcus mutans. These bacteria are beta-hemolytic cocci and known to cause several diseases in humans, including mild conditions like scarlet fever, impetigo, strep throat, caries and cellulitis, and more severe illnesses like necrotizing fasciitis (flesh eating disease) and toxic shock syndrome (TSS).

49Streptococcus pyogenes comprises the considerable majority of Group A Streptococci (GAS); it is a pathogen responsible for several human diseases such as pharyngitis, scarlet fever, toxic shock syndrome, pneumonia and others. Recent studies have shown that GAS was able to resist the action of several human antimicrobial peptides such as cathelicidin, LL-37 and the α-defensin (HNP-1). The surface exposed M-protein is used to classify the bacterium into different serotypes. have shown that the N-terminal portion of M-protein can interact with LL-37, preventing its action on the bacterium membrane.

50Streptococcus mutans is an important pathogen that colonizes the human oral cavity being the most important caries agent. Interestingly, several S. mutans strains have been described as resistant to salivary AMPs and bacitracin.

51A study by compared S. mutans strains isolated from 60 children divided into two groups (caries-free and caries-active) and they found that strains isolated from the caries-active group were significantly more resistant to salivary AMPs such as LL-37, α-defensins and β-defensins, in comparison to caries-free strains. Their analysis also correlates this resistance to an ecological advantage over the less resistant strains, which reinforces the importance of AMPs in controlling S. mutans colonization.

52Since most AMPs present cationic nature, the negative charge of the bacterial surface is important for the bactericidal activity of these molecules. showed that the D-alanylation (regulated by the operon dlt (DltABCD)) of S. pyogenes lipoteichoic acid is related with resistance to cationic AMPs, lysozyme and low pH, and it was also associated with an increased survival against neutrophil killing; this phenomenon is due to the increase of positive surface charge caused by the D-alanylation on the cell membrane. In another study, using a knockout strain for the dltABCD operon found that the DltA mutant displayed a drastic reduction in the expression of M protein and SIC (Serum Inhibitor of Complement), showing that the operon dlt (DltABCD), specifically the dltA gene regulates the expression of genes involved in AMP resistance.

53A study published by investigated the mechanisms that allow S. mutans to resist bacitracin; they found that mutant strains lacking the rgp locus (a six gene operon) presented up to five times more sensibility to bacitracin than the wild type counterpart. A possible mechanism to explain this sensitiveness is the fact that the rgp locus is involved in the synthesis of rhamnose-glucose polysaccharide (RGP), a cell wall component; mutations affecting this process render the bacterium more sensitive to bacitracin.

54Streptococcus pyogenes is able to limit LL-37 action through degradation by the cysteine proteinase, SpeB. In presence of the inhibitor E64 (which inhibited the cysteine proteinase) the bacterium’s ability to degrade LL-37 was hampered, making it more susceptible to this CAMP. This effect highlights the importance of proteinase SpeB in LL-37 degradation.

55Similarly, to previously described for Enterococci, S. pyogenes secretes proteases that are able to cleave proteoglycans containing dermatan sulfate, releasing it to the extracellular space. The extracellular dermatan sulfate was able to neutralize neutrophil-derived alpha-defensin, protecting the bacteria from its bactericidal activity.

56M-protein is the most studied protein in S. pyogenes; variations in M-protein sequence are used to classify the bacterium into different serotypes. A study by, showed that the M protein type 1 protects the bacterium from killing by cathelicidins LL-37 (human) and mCRAMP (mouse). The proposed mechanism involves M1 binding to and trapping the cathelicidin before it can reach the cell wall. They also showed that this protection is type specific once M protein type 49 did not protects the bacterium the same extension of M1, moreover, they found that strains isolated from invasive diseases patients were more resistant to LL-37 action than the strains isolated from asymptomatic patients.

57Another strategy employed by S. pyogenes to resist AMP attack is the Serum Inhibitor of Complement (SIC). This protein was initially identified as a virulence factor protecting the bacterium against killing by the complement system membrane attack complex. Further studies from the same group showed that SIC is important for bacterium full virulence, once it is able to bind to defensins and LL-37, protecting the bacterium against these molecules.

58Streptococcus mutans express the ABC transporter, mbr, an operon composed by 4 genes. Mutant strains that do not express the full transporter were 100 to 120-fold more sensitive to bacitracin than the wild type strain. A more recent study from the same group, analyzed the transcriptome of the bacterium after exposure to bacitracin. They found 8 genes (SMU.302, SMU.862, SMU.863, SMU.864, mbrA, mbrB, SMU.1479, SMU.1856c) that were upregulated upon AMP challenge; of those, the MbrC protein acts as a transcriptional regulator for MbrA and MbrB–which are part of the ABC transporter and are required for bacitracin resistance–and it also controls the expression of SMU.863 and SMU.864, also described as ABC transporters involved in bacitracin resistance by S. mutans.

59The S. mutans bceABRS operon encodes an ABC transporter (BceAB) and a two-component system BceRS. The entire four-component system was shown to be important for protection against bacitracin, defensins (α and β), LL-37 and histatin. In contrast with wild type S. mutans, mutant strains lacking each bceABRS gene failed to form biofilms in response to a sub-inhibitory concentration of β-defensin. This data suggest that BceABRS also acts as a sensor, promoting a switch to an AMP resistant phenotype upon challenge.

60Streptococcus agalactiae, also referred to as Group B Streptococci (GBS), is an opportunistic pathogen that colonizes the gastrointestinal, genitourinary tracts and, in women, the vaginal mucosa. The biggest concern regarding infections with GBS is in pregnant women, because it can be transmitted vertically and results in serious neonatal consequences, causing several diseases to the newborn, such as meningitis, sepsis and pneumonia.

61The incidence of infections by S. agalactiae is twice as high in pregnant women when compared to non-pregnant women. Most GBS infections occur during labor, but there is also a chance of infection after delivery. In the United States, GBS infection rates range from 0.1 to 0.8 per 1,000 childbirths. Worldwide, the rates in pregnant women are 0.38 per 1,000 childbirths, with 0.2 in 1000 mortality rate. GBS infection is also associated with an increased chance of premature delivery. Around the world, premature birth is an important contribution to the death of newborns; approximately 10% of deaths in neonates are caused by GBS infection.

62In S. agalactiae, the dlt operon is essential for resistance against AMPs. Deletion of dltA hinders bacterial survival ability in vivo and reduces the resistance to AMPs, possibly due to an increased interaction with the peptide. Interestingly, the D-alanylation of the membrane seems to induce resistance by enhancing cell envelope strength rather than the interference with the ionic charge of the membrane.

63Streptococcus agalactiae is intrinsically resistant to nisin via NSR or SaNSR, a nisin-specific enzyme that cleaves and hinders the activity of the peptide. It is expressed by the nsr operon with other lantibiotic resistance genes, such as nsrFP and nsrRK. However, modified nisin molecules were able to maintain activity against strains possessing SaNSR. Another mode of escaping the degrading activity of AMPs is via inhibitory molecules capable of binding to the nisin site of SaNSR. A phosphoglycerate kinase of GBS was also identified to participate in AMP resistance. Though the mechanism is unknown, it is supposed to include direct binding of the peptides.

64NsrFP is an ABC transporter which exports nisin to the extracellular medium. The transporter binds to the N-terminal portion of the peptide and releases it, preventing cell death, even in absence of the two-component regulator NsrRK.

65In S. sanguinis, a study involving multiple gene screening reported a role for sag1003 in AMP resistance against nisin and bacitracin. The gene is predicted to be an efflux pump against AMPs and a transposon-induced mutagenesis caused a higher sensitivity against both AMPs in a plate-based minimum inhibitory concentration (MIC) assay.

66Streptococcus agalactiae pili are important against host defense mechanisms, such as AMPs. The sequestration of AMPs by pili prevents the interaction with the membrane targets. Strains lacking pilB, one of the pilus subunit proteins, were more sensitive to AMPs and less virulent overall, supposedly by virtue of resistance against LL-37, mCRAMP and polymyxin B. Heterologous overexpression of PilB from S. agalactiae in L. lactis showed similar results.

67The bceRSAB is a detoxification system in GBS, regulating the gene expression against AMPs, such as dltA, promoting resistance. Strains lacking the regulator BceR showed an increased susceptibility against bacitracin and LL-37 and reduced overall virulence.

68The insertion of an inactivation transposon in sag1003 induced a reduction of phosphoglycerate kinase in the cell wall.

69The two-component regulator NsrRK is responsible for the transcriptional control of the NSR pathway (nsr and nsrFP) in L. lactis strains capable of synthesizing nisin. In GBS, a very similar nsr operon was described, indicating the possibility of an analogous system.

70identified a surface-associated penicillin-binding protein called PBP1a, which is encoded by the ponA gene. A mutant ΔponA strain was more susceptible to AMPs from cathelicidin and defensin families, but the exact mechanism involved in this protection is still unknown.

71Streptococcus pneumoniae (pneumococcus) is responsible for around 1 million deaths worldwide every year, and an increasing drug resistance case reporting. It is the main causative agent in community acquired bacterial pneumonia, and it can also cause otitis media, conjunctivitis, sinusitis and more severe diseases like meningitis and bacteremia.

72Pneumococci are frequent colonizers of the upper respiratory tract, and a single person may be colonized with multiple strains concomitantly for months. Asymptomatic carriers are also the main source of pneumococcal transmission. In this highly colonized niche, AMP resistance confers an important competitive advantage both inter and intra species. Pneumococci display a vast number of adaptations that promote increased AMP resistance, from envelope modifications to AMP sequestration, as described next.

73A vast majority of clinically relevant pneumococcal isolates are covered by a thick polysaccharide capsule with variable structure, which protects the bacterium from host immune defenses. Based on their high immunogenicity and protective efficacy, capsular polysaccharides comprise the basis of the current pneumococcal vaccines, alone or in fusion with carrier proteins.

74Variations in capsule polysaccharide locus determine the classification of pneumococci in over 95 different serotypes. These include mainly negative structures, with a few being neutral or positive. Negatively charged free capsular polysaccharides (but not neutral or positive ones) have displayed a role in preventing AMP attack. These purified anionic CPS were able to increase the resistance of non-encapsulated mutant pneumococci to HNP-1 and polymyxin B, an effect that was abrogated when the CPSs lost their negative charge through reaction with polycations. One proposed mechanism is that exposure to antimicrobial peptides triggers CPS release, which trap the AMPs and shield the bacterium. This capsule shedding has been demonstrated to occur in vivo, thus comprising a potential strategy to prevent AMP-mediated killing. Capsule shedding can be triggered by autolysin (LytA) activity, promoting bacterial resistance to LL-37 and favoring colonization.

75Surface-attached capsular polysaccharides, on the other hand, have shown the opposite effect, rendering the bacteria more susceptible to AMP action, in comparison with non-encapsulated isogenic mutants. This effect was observed with different capsular types, including CPS 2, 4, 9V and 19F, and the zwitterionic serotype 1. As shown for other Gram-positive bacteria, D-alanylation of teichoic acids in non-encapsulated pneumococci results in increased resistance against killing by neutrophil extracellular trap (NET)-derived components. This effect is aided by surface proteins, like the choline binding protein LytA and PgdE, which contribute to reduce the surface negative charge (discussed further). In that sense, the presence of capsule could mask the underlying protective mechanisms against AMPs. This apparent detrimental effect of capsule production over pneumococcal sensitivity to AMPs is possibly overcome by the capsule shedding as previously discussed, and also by its ability to protect the bacterium against mucus and phagocytic cell repulsion. Furthermore, the effect may not be applicable to all capsular serotypes; great variations in carriage, invasiveness and prevalence exist among capsule types, which have been associated with variations in surface net charge. In that sense, the investigation of AMP resistance in a higher number of pneumococcal serotypes may provide new insights into the role of surface CPS on AMP resistance. For instance, type 4 TIGR4 and its isogenic capsule-negative mutant have shown increased sensibility to CXCL10, LL-37, and nisin, when compared with the type 2 strain, D39.

76Another study has shown that non-encapsulated pneumococci are more resistant to neutrophil proteases, elastase and cathepsin G–a feature that also contributes to the ability to colonize the nasopharynx.

77Cell wall modifications by the dlt operon have also been shown to promote resistance against nisin and gallidermin in pneumococci, an effect that was consistent with an increased release of D-alanine upon hydrolysis in wild type versus dltA-negative mutant stains.

78Pneumococci express two enzymes, PgdA and Adr, that modify peptidoglycans on the bacterial cell wall. PgdA is a N-acetylglucosamine deacetylase, while Adr is an O-acetyl transferase that acetylates muramic acid residues on the peptidoglycan backbone. Double mutant strains unable to perform these modifications displayed lower ability to colonize lysozyme-sufficient mice, but behaved similarly, to wild type pneumococci in mice lacking lysozyme production. In contrast, mutants in only one of the molecules colonized mice more efficiently than the wild type strain, in both Lys-producing and Lys-deficient mice. Taken together, these results indicate that the ability to limit lysozyme attack by modifying the cell wall contributes to successful colonization of the host.

79Studies from our group and others have reported a role for pneumococcal surface protein A (PspA) in bacterial resistance to AMPs. PspA is an exposed virulence factor with structural and serological variability that has been successfully evaluated as a vaccine candidate in different infection models. It prevents complement activation/deposition on the pneumococcal surface, limiting bacterial uptake by phagocytes.

80Pneumococcal surface protein A can bind to and prevent the lytic action of lactoferrin. Furthermore, anti-PspA antibodies induced by vaccination were able to enhance the bactericidal effect of apolactoferrin (the iron-free form of the molecule) by blocking PspA interaction with that protein. This protective effect of PspA over pneumococci was diminished when lactoferrin was combined with lysozyme. This set of data suggests PspA is able to prevent the lytic action of cationic peptides against pneumococci, possibly by binding to these molecules through their active sites. This interaction has been demonstrated for lactoferrin.

81Pneumococcal surface protein A has also been shown to interfere with the bactericidal activity of NETs. Mutants lacking PspA were more susceptible to trapping by NETs, an effect that was dependent on PspA type. In addition, incubation with anti-PspA antibodies promoted NET formation. Taken together, the data indicates that PspA is able to directly prevent killing by AMPs, and also to limit the bactericidal mechanisms of neutrophils.

82Pneumococci express and efflux pump, MefE/Mel, which confers resistance to macrolides. mfE expression is induced upon bacterial incubation in presence of LL-37. In consequence, pneumococci develop resistance to LL-37 and erythromycin in vitro.

83A second, MacAB-like efflux pump described in S. pneumoniae, comprised by the spr0693-spr0694-spr0695 operon, is also involved in resistance against antimicrobial peptides and antibiotics, like LL-37, nisin and bacitracin.

84The oligopeptide import ABC transport system Opp (AmiACDEF) has been implicated in resistance against CXCL10, a chemokine with antimicrobial activity against several pathogens. In that work, mutant strains lacking the permease were less susceptible to CXCL10 and nisin, when compared with the parent D39 strain. Although the precise mechanism responsible for this effect is not fully understood, it is known that AmiA-F has additional pleiotropic roles in pneumococcal physiology, quorum sensing, and virulence.

85Cell wall modifications in pneumococci can be triggered by AMPs. Treatment with lysozyme leads to upregulation of the dlt locus through the CiaRH sensoring system, resulting in lipoteichoic acid (LTA) modifications and increased inflammatory responses, which in that case contributed to bacterial shedding and transmission. Thus, D-alanylation of the cell wall – a mechanism of AMP resistance shared among different Gram-positive microbes–can be induced, in pneumococci, by treatment with antimicrobial proteins that target the bacterial cell wall.

86Incubation with LL-37 can also trigger an adaptive response in pneumococci. The transcriptome analysis of pneumococci treated with LL-37 revealed a profound effect on the bacterial genome, with 10% of the genes displaying an altered expression upon challenge. The up-regulated genes included those involved in cell wall biosynthesis (dlt), bacteriocin production, virulence (such as the proteases HtrA e PrtA) and bacteriocin production, as well as transcriptional regulators and putative ABC transporters. Interestingly, the serino-protease HtrA is also involved in resistance against other environmental stressors, like high temperature and oxidative stress. The choline binding protein PspA and LysM protein (SP 0107)–predicted to be involved in cell wall metabolism–were down regulated in presence of LL-37. Interestingly, LL-37 had a much more dramatic effect on pneumococcal gene expression patterns, when compared with bacterial-derived AMPs that act on the same bacterial targets (nisin and bacitracin). Furthermore, mutant strains lacking these genes revealed an increased susceptibility to treatment with LL-37, confirming the employment of multiple defense strategies against AMPs in pneumococci. A more recent study evaluating the proteome of pneumococci treated with LL-37 has also reported a large number of proteins with altered abundance, including transporters, proteins involved in gene regulation and cell wall modification, virulence factors (such as Pht family) and the protease HtrA. This result suggests that multiple mechanisms cooperate in pneumococcal response to AMPs.

87A study investigating the susceptibility of multiple pneumococcal isolates – both clinical and from carriage–to LL-37 and HNP-1 found great variations in AMP resistance, with no correlation with AMP or capsule type, although clinical isolates were, in general, more susceptible than were carriage isolates. Furthermore, the study reported that AMP challenge could affect bacterial fitness in competitive assays. This result suggests a role for AMPs in driving intraspecific competition among pneumococci in the nasopharynx, contributing the bacterial genetic diversity in this niche.

88Antimicrobial peptides are central players in the innate immune defense against pathogenic bacteria. Unsurprisingly, microbes have developed several strategies to overcome AMP activity, which allow them to efficiently colonize/invade the host. The present review summarizes the strategies adopted by Gram-positive pathogenic bacteria to resist AMP action. Some of these mechanisms, like cell wall modifications, are shared by several pathogens, highlighting their pivotal contribution to bacterial survival within the host. Other factors such as surface proteins and virulence factors are microbe-specific, revealing a myriad of adaptations that comprise the bacterial arsenal against AMPs.

89The alarming increase in antibiotic resistance has prompted the search for alternative treatment options. In this scenario, AMPs emerge as a promising strategy to control bacterial infections. This rationale is reinforced by the demonstration that antibiotic resistance in bacteria usually correlates with a collateral sensitivity to AMPs.

90Several approaches employing AMPs have been tested with encouraging results. The use of AMP combinations is of particular interest, since these molecules can potentiate each other’s action, and also improve the therapeutic efficacy of conventional antibiotics, through synergistic interactions, This represents an excellent strategy to slow down or minimize bacterial resistance development. In that sense, a better comprehension of the mechanisms employed by bacteria to resist AMP action is pivotal for the development of more effective therapeutic strategies. Furthermore, since many bacterial molecules involved in AMP resistance are important virulence factors, the present review presents numerous potential targets for vaccine development, and also contributes to elucidate the mechanisms driving intra-and interspecies competition within the host.

91LA, TC, BM, MC, NW, LN, MG, and MD drafted the manuscript. LA, TC, and MD revised the manuscript. LA and MC drafted the table. LA, MC, and MD produced the Figure. All authors read and approved the final manuscript. None of the authors have any competing interests.

92The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.