Short history of discovery - Responses of pseudomonas aeruginosa and other eskape pathogens to

Introduction

1.1 Short history of discovery

The introduction of antibiotics has been the most important step in the fight against bacterial infections. In 1907, Ehrlich and Bertheim synthesized the first antimicrobial agents known as the sulpha drugs or sulfonamides. These molecules are made of a sulfonamide group and affect the folate biosynthesis pathway by inhibiting the dihydropteroate synthetase (DHPS) enzyme (Williams 2009). Nevertheless, it is considered that Domagk discovered the first sulfonamide “Prontosil” that was used in clinical settings and earned him a Nobel Prize in 1939 (Otten 1986). In 1928, sir Alexander Fleming discovered penicillin (Fleming 2001) and its active compound was further purified by Florey and Chain (Ligon 2004). This discovery earned them the Nobel Prize in 1945 and is admitted as the discovery of the first authentic antibiotic when referring to the definition of Waksman: an antibiotic is a molecule synthesized or derived from microorganisms that kills other microorganisms or inhibits their growth (Waksman 1947). The introduction of penicillin enabled the treatment of disease such as streptococcal infections that were until then untreatable. The progressive discovery of different classes of antibiotics occurred during the “golden era of antibiotics” between 1940-1960’s (Fig. 1). Considerable research and industrialization followed these discoveries.

Figure 1. Timeline of antibiotics development and resistance appearance. Top panel illustrates the introduction of main antibiotics classes and bottom panel illustrates when resistance to these classes of antibiotics was observed. Adapted from (Clatworthy, Pierson and Hung 2007).

12 1.2 Spectrum of activity

Based on their spectrum of activity, antibiotics can be classified in two groups: broad spectrum and narrow spectrum. Broad spectrum antibiotics are useful to treat both Gram-positive and Gram-negative bacteria. Narrow spectrum antibiotics are used when a single pathogen is responsible for an infection and decrease the side effects on the bacterial flora of the patient (Acar 1997, Willing, Russell and Finlay 2011, Blaser 2011). One such example can be found among the cephalosporin antibiotics. Ceftazidime is a 3^rd generation cephalosporin with broad spectrum, whereas Cefuroxime, which is of 2^nd generation, has a narrower spectrum (O'Callaghan 1986, Perry and Brogden 1996). The spectrum of activity relies on structural membrane differences between Gram-negative and Gram-positive bacteria (Fig. 2), as well as the presence of intrinsic resistance mechanisms.

Even if most antibiotics cross the cell membrane through passive transport using porins or other transporters, Gram-negative bacteria remain more challenging to kill as they have an outer membrane (Tommasi et al. 2015). The Gram-negative bacteria are characterized by both an inner and outer membrane composed of a bilayer of phospholipids, and a thin peptidoglycan (cell wall) linked to the membrane through lipoproteins in the periplasmic space. The outer membrane of Gram-negative bacteria also contains lipopolysaccharides (LPS). On the other hand, Gram-positive bacteria only have an inner membrane and a thick peptidoglycan, containing lipoteichoic acids and teichoic acids. negative and Gram-positive bacteria have common structures that include flagella and for some bacteria a capsule, which is made of glycoproteins or proteins that can provide an additional layer of protection (Cabeen and Jacobs-Wagner 2005). Both Gram-positive and Gram-negative organisms also express different drug efflux systems reducing the intracellular concentration of antibiotics (Poole 2005).

Figure 2. Gram-negative and Gram-positive bacterial membranes. Adapted from (Brown et al. 2015).

Antibiotics can be either bactericidal or bacteriostatic. A bactericidal antibiotic kills bacteria whereas a bacteriostatic antibiotic inhibits the bacterial growth and the infection needs to be cleared by the host’s immune system. It should be noted that these definitions apply at the level of the organism and under in vitro conditions, but in a clinical context they become arbitrary (Pankey and Sabath 2004). Some antibiotics can exert a bactericidal effect against some bacteria and a bacteriostatic effect against others such as chloramphenicol, which has been shown in vitro to be bactericidal against S. pneumoniae and bacteriostatic against S.

aureus (Rahal and Simberkoff 1979).

1.3 Antibiotic classes and targets

Antibiotics can be classified according to their mode of action. Only few bacterial pathways can be targeted due to the fact that the antibiotics should not affect the eukaryotic cells and consequently, only pathways that are specific to the bacteria can be targeted. Based on their targets antibiotic classes include 1) Inhibitors of transcription, 2) Inhibitors of DNA replication, 3) Inhibitors of protein synthesis, 4) Inhibitors of the folic acid synthesis, 5) Inhibitors of the cell wall synthesis (Fig. 3). Most of these molecules were extracted and purified from natural producers (bacteria of fungi), only few are entirely synthetic. Usually new molecules are developed based on the structure of already described antibiotics, but with some modifications or an increased affinity for the same targets (Nathan 2004, Fischbach and Walsh 2009). One such example of this development are the cephalosporins, a class of b-lactams.

They are separated into 1^st, 2^nd, 3^rd, 4^th and 5^th generation according to changes of their structure and the spectrum of activity (Fischbach and Walsh 2009, Bassetti et al. 2013).

The rifamycins, like Rifampicin, target the transcription. They bind to the DNA-dependent RNA polymerase holoenzyme before the DNA unwinds to keep it closed and inhibit RNA synthesis (Hartmann et al. 1967, Naryshkina et al. 2001).

The quinolones and fluoroquinolones, which are derived from quinolones by addition of a fluorine atom like ciprofloxacin, inhibit the DNA gyrase (topoisomerase II) and DNA topoisomerase IV. These enzymes are involved in DNA topology changes, a step required during DNA synthesis. After cleavage and before re-ligation, these enzymes introduce positive (topoisomerase IV) and negative (topoisomerase II) supercoils in the DNA. Fluoroquinolones bind to topoisomerase II and block the DNA re-ligation (Redgrave et al. 2014).

Ribosomal activity is required in translation and involves the 70S complex, which is composed of the two subunits 30S and 50S. Some antibiotics, like aminoglycosides and tetracyclines, inhibit the 30S subunit. Aminoglycosides bind to the 16S rRNA and alter the mRNA-tRNA complex or inhibit the translocation of peptidyl-tRNA. This leads to incomplete translation termination (Davis 1987). Binding to the 16S rRNA of the 30S subunit by tetracyclines prevents the tRNA binding to the A site (Chopra and Roberts 2001). Other antibiotics, like macrolides and chloramphenicol, inhibit the 50S unit by binding nearby the peptidyl transferase center.

Premature protein biosynthesis termination occurs through the dissociation of the ribosome and the peptidyl tRNA (Egorov, Ulyashova and Rubtsova 2018, Menninger and Otto 1982).

The synthesis of folic acid is an essential pathway as organisms require the folate cofactor, which is involved in nucleic acids and amino acids biosynthesis. The dihydropteroic acid is synthesized from para-amino-benzoic acid (PABA) and pteridine by the dihydropteroate synthetase (DHPS) enzyme. Dihydrofolate synthetase then synthesizes dihydrofolic acid from dihydropteroic acid. Dihydrofolic acid is used by dihydrofolate reductase (DHFR) to synthesize tetrahydrofolic acid (Bermingham and Derrick 2002). This pathway is inhibited by the sulfa drugs such as sulfamethoxazole, which inhibits the DHPS enzyme, and trimethoprim, which inhibits the DHFR enzyme (Masters et al. 2003). These molecules also target the eukaryotic enzymes, however the concentrations required to inhibit these enzymes are higher than those used against bacteria leaving a therapeutic window for sulfonamides.

The cell wall synthesis inhibitors include β-lactams, such as carbapenem, penicillin, monobactam and cephalosporin that target the penicillin binding proteins (PBPs) (Holten and

Onusko 2000). In the synthesis of peptidoglycan (PG), the glycosyltransferases (GTases) polymerize the glycan chains, whereas the DD-transpeptidases (DD-TPases), which are also called PBPs are involved in the cross-linking of the peptides. The β-lactams covalently bind to the active site of PBPs and inhibit the synthesis of PG (Typas et al. 2011). In bacteria, various PBPs are involved in the cell cycle depending on which step they act and the localization (Randich and Brun 2015). Due to strong affinity, the β-lactams are most of the time linked with specific PBPs.

Figure 3. Antibiotic targets. (Madigan, Madigan and Brock 2009).

1.4 Antibiotics resistance

The use of antibiotics is an essential part of modern medicine that has improved health conditions and life expectation (Piddock 2012). It is also of great interest in agriculture and animal production to prevent and treat bacterial diseases (Gustafson and Bowen 1997).

Nevertheless, the extensive use of antibiotics has promoted the selection and proliferation of resistant bacteria (2017). Antibiotic resistance leads to approximately 700’000 deaths per year and is estimated to cause up to ten million deaths per year by 2050 (Blair et al. 2015).

Antibiotic resistance has become a major concern and there is an urgent need to develop novel antibiotics. The emergence of antibiotic resistance is a natural event first observed in

the 1940s and which has spread worldwide. For example, the β-lactamase enzymes, which deactivate the β-lactam antibiotics by hydrolysis, have been described before the introduction of penicillin in 1942 (Spellberg et al. 2011). Historically, the appearance of resistance to a particular antibiotic could be observed shortly after it was introduced (Fig. 1). Antibiotic resistance is a phenomenon that is encoded genetically and can be divided in three categories:

intrinsic, acquired and adaptive resistance. Intrinsic resistance includes all the inherent characteristics of a microorganism that can inhibit the action of an antibiotic. The outer membrane with reduced permeability as in Pseudomonas aeruginosa and Acinetobacter baumannii, or the efflux pumps encoded by bacteria to eject antibiotic from the cell, are examples of intrinsic resistance mechanisms (Lewis 2013). In acquired resistance, the bacteria, which are originally susceptible, become resistant through the incorporation of genetic material (integrons, transposons, plasmids, etc.) or mutations. In adaptive resistance, antibiotic susceptible bacteria respond to the presence of an antibiotic, or a stress condition, by modifying transiently their gene expression leading to target modifications or efflux pump overexpression (Fernández and Hancock 2012).

2. Antimicrobial peptides against Gram-positive bacteria

Since the emergence of antibiotic resistance, the search for alternate antimicrobial drugs has become of high priority. Bacteria that are not susceptible to at least one antimicrobial in more than two antibiotic classes are considered as multi-drug resistant (MDR) strains (Magiorakos et al. 2012). Among Gram-positive bacteria, Staphylococcus aureus is one of the most prevalent MDR strains that causes both community- and hospital-acquired infections (Tong et al. 2015). Methicillin-resistant S. aureus (MRSA) are a great concern as they are difficult to treat (Turner et al. 2019). Enterococcus species such as Enterococcus faecium and Enteroccocus faecalis are also another concern as they cause various infections and are resistant to a broad range of antibiotics (Jabbari Shiadeh et al. 2019). Among enterococcal infections, 85-90% are due to E. faecalis and 5-10% to E. faecium. Vancomycin-resistant Enterococci (VRE) can be found among both species (Ho et al. 2013). Streptococcus pneumoniae is another Gram-positive pathogen causing community-acquired infections such as pneumonia and meningitis. Some strains of S. pneumoniae have been described to be resistant to penicillin (Thummeepak et al. 2015). Antimicrobial peptides (AMPs) were suggested as promising candidates because of their potentiality to fight multi-drug resistant

bacteria (Seyfi et al. 2020). In this section, antimicrobial peptides available against Gram-positive bacteria are reviewed.

2.1 Structure and mode of action 2.1.1 Glycopeptides

Vancomycin is the first glycopeptide antibiotic isolated from Amycolatopsis orientalis in 1955 (McCormick et al. 1955). It consists of a heptapeptide core that is common to all glycopeptides (Fig. 4). Vancomycin binds to the terminal D-Alanine-D-Alanine (D-Ala-D-Ala) of peptidoglycan precursors through five hydrogen bonds, and inhibits transglycosylation and transpeptidation preventing thereby cell wall synthesis (Fig. 5). Vancomycin is used to treat Gram-positive bacterial infections and especially MRSA infections (Courvalin 2006). It displays a good activity against staphylococci, enterococci and streptococci with minimum inhibitory concentration (MIC) of 1 µg/mL, 2 µg/mL and 0.25 µg/mL, respectively (Table 1).

Figure 4. Structures and characteristics of AMPs against Gram-positive bacteria. Structures of glycopeptides, semi-synthetic glycopeptides and antimicrobial peptides are represented with their molecular weight (M.W.), half-life (T1/2) and mode of action (MoA) as well as the number of positive charges in red. Adapted from (Chen and Lu 2020).

Teicoplanin is a glycopeptide similar to vancomycin isolated from Actinoplanes teichomyceticus with a molecular weight varying between 1564 and 1907 g/mol, and carrying one positive charge (Fig. 4). This molecule is made of a mix of several components, five major (teicoplanin A2-1 to A2-5) and four minor (teicoplanin RS-1 to RS-4), and they all possess the same glycopeptide core but the length and conformation of the side chains are different (Barna et al. 1984). Teicoplanin differs from vancomycin by the presence of a hydrophobic acyl side chain (Nicolaou et al. 1999). As for vancomycin, the mode of action of teicoplanin involves inhibition of cell wall synthesis (Khamesipour et al. 2015).

Figure 5. Mode of action of glycopeptides. The binding of the glycopeptide to terminal D-Ala residues of peptidoglycan precursors (A) prevents their interaction with cell wall cross-linking enzymes (B) and results in inhibition of cell wall synthesis.

2.1.2 Semi-synthetic glycopeptides

Telavancin is a semi-synthetic lipoglycopeptide derived from vancomycin. It differs from vancomycin by the presence of a hydrophilic group and a hydrophobic side chain on the vancosamine sugar (Fig. 4). Like vancomycin, telavancin inhibits cell wall synthesis by binding to the terminal D-Ala-D-Ala of peptidoglycan precursors (Attwood and LaPlante 2007). The presence of the hydrophilic group confers to telavancin a second mechanism of action. It can interact with lipid II and disrupt the membrane by causing depolarization (Higgins et al. 2005).

Dalbavancin is a second generation semi-synthetic lipoglycopeptide and a derivative of the A-40926 antibiotic, which is isolated from Nonmuria spp. and has a similar structure to

teicoplanin (Bailey and Summers 2008, Zhanel et al. 2008) (Fig. 4). The modifications made to the structure by addition of a lipophilic side chain that is not present in teicoplanin increase its half-life and activity against S. aureus. The dalbavancin mode of action also leads to inhibition of cell wall synthesis. The lipophilic side chain increases the affinity of binding to the terminal D-Ala-D-Ala of peptidoglycan precursors (Zhanel et al. 2008, Kim, Kuti and Nicolau 2007).

Oritavancin is derived from chloroeremomycin, which is similar to vancomycin and produced by Amicolatopsis orientalis (Cornaglia and Rossolini 2009). Like vancomycin, it is made of a heptapeptide core but has in addition a lipophilic side chain that provides a longer half-life (Fig. 4). As other glycopeptides, oritavancin binds to the terminal D-Ala-D-Ala of peptidoglycan precursors and inhibits cell wall synthesis. Furthermore, the hydrophobic side chain of oritavancin allows disruption of the membrane (Allen and Nicas 2003).

Table 1. Antimicrobial activity against Gram-positive bacteria.

MIC (mg/L)

Vancomycin Teicoplanin Dalbavancin Oritavancin Daptomycin Gramicidin

S. aureus 1 0.5 0.06 0.12 0.5 4

Daptomycin is a cyclic lipopeptide of 13 amino acids introduced in 2003 and isolated from Streptomyces roseosporus (Debono et al. 1987) (Fig. 4). The mode of action of daptomycin involves the formation of a complex with Ca²⁺ that inserts into the cell membrane of Gram-positive bacteria. Aggregation of daptomycin results in the depolarization of the membrane and subsequently the loss of cell content (Tran, Munita and Arias 2015) (Fig. 6).

Figure 6. Mode of action of daptomycin. Daptomycin get inserted into the cell membrane in a Ca²⁺

dependent manner and results in loss of intracellular components through membrane depolarization.

Adapted from (Bionda, Pitteloud and Cudic 2013).

Gramicidin was first isolated from the soil bacteria Bacillus brevis although its function in these bacteria is unknow and has a structure made of L- and D-amino acids (Sarges and Witkop 1965) (Fig. 4). Two types can be found, the linear pentadecapeptide Gramicidin D and the Gramicidin S, which is a cyclic decapeptide. The activity of Gramicidin D occurs by interaction with the bacterial membrane through monovalent cations resulting in increased permeability.

Gramicidin S acts by disrupting the inner content of the cell (Conti et al. 1997). Even though Gramicidin is among the most effective peptides, its use as an antibiotic peptide is restricted due to a high hemolytic activity (Mogi and Kita 2009).

2.2 Mechanism of resistance

Resistance to vancomycin involves modification of the peptidoglycan synthesis pathway by replacing the terminal D-Ala-D-Ala of peptidoglycan precursors by D-Ala-D-Lac, which prevents binding of vancomycin, and occurs through the vancomycin resistance (van) operon.

In enterococci, this operon can be carried chromosomally or on a plasmid. This operon includes the two component regulatory system VanS/R with vanS being a membrane histidine kinase and vanR the response regulator, the D-lactate dehydrogenase gene vanH, a variable ligase vanA or vanB, the D-Ala-D-Ala dipeptidase gene vanX (Fig. 7). Activation of VanS by vancomycin results in the induction of vanHAX expression through VanR. VanH provides D-Lac from the reduction of pyruvate, whereas free D-Ala are provided through VanX activity, which are incorporated by VanA to produce Lac pentapeptides. Furthermore, the D-Ala-D-Ala that is constitutively synthesized is removed by VanX (Faron, Ledeboer and Buchan 2016).

The replacement by D-Lac results in a decrease of affinity to vancomycin of almost 1000-fold (Courvalin 2006).

Figure 7. Vancomycin resistance (van) operon. The VanSR two-component regulatory system responds to vancomycin that leads to expression of vanH, vanA or vanB, and vanX downstream genes.

The D,D-dipeptidase VanX cleaves repeats of D-Ala-D-Ala and thereby provides free D-Ala for VanA/B, whereas the D-hydroxyacid dehydrogenase VanX provides D-Lac through reduction of pyruvate. VanA and VanB ligases enable the production of D-Ala-D-Lac pentapeptides with low affinity for vancomycin.

The D,D-carboxypeptidase VanY cleaves the D-Ala terminal peptide, which results in reduction of pentapeptides with high affinity to vancomycin. VanZ is involved in teicoplanin resistance through unknown mechanism and is present in strains carrying vanA. Adapted from (Faron et al. 2016).

Enterococci bearing the vanA gene show a high level of vancomycin resistance and are the predominant vancomycin-resistant Enterococcus (VRE) among E. faecium and E. faecalis, whereas strains carrying vanB gene display a moderate level of resistance (Faron et al. 2016).

In S. aureus, two phenotypes of decreased susceptibility to vancomycin can be distinguished;

vancomycin intermediate-resistant S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA). VRSA strains are less frequent and acquired vanA operon from a VRE E. faecalis. VISA strains appeared from vancomycin-susceptible S. aureus (VSSA) by accumulation of mutations that affect cell-envelope homeostasis with the most involved genes being walkR, vraRS and graRS. This results in a broader cell wall with increased free D-Ala-D-Ala dipeptides that reduce cross-linking (McGuinness, Malachowa and DeLeo 2017). Similarly, to vancomycin, teicoplanin, dalbavancin and telavancin resistance occurs by modification of the peptidoglycan precursors and involves the expression of the van operon, whereas resistance to oritavancin has not been observed in clinical isolates (Rosenthal et al. 2018).

Table 2. Genes involved in mechanisms of AMP resistance in Gram-positive bacteria.

Function E. faecalis E. faecium S. aureus

Daptomycin

mprF incorporates positive charged amino

acid lysine to PG NI NI Repulsion

dlt operon incorporates D-alanine into cell-wall

teichoic acids NI Repulsion Repulsion

cls cardiolipin synthase Diversion Repulsion Repulsion

lia locus 3-component regulatory syst. as part

of cell envelope stress response Diversion NI NI gdpD glycerophosphoryl diester

phosphodiesterase Diversion NI NI

Vancomycin, Teicoplanin, Telavancin, Dalbavancin van operon production of D-Ala-D-Lac

pentapeptides ✓ ✓ ✓

NI, not involved.

Two mechanisms have been proposed for daptomycin resistance. The first one is diversion of daptomycin from the preferential binding site. The anionic phospholipids, such as cardiolipin, are redistributed away from the septum, resulting in inadequate binding of daptomycin (Fig.

8A). This mechanism is found in E. faecalis and is associated with expression of several genes (Table 2). cls and gdpD encode for enzymes involved in the metabolism of phospholipids, a cardiolipin synthase and a phosphodiesterase, respectively, whereas the liaFSR locus encodes a three-component regulatory system involved in cell-envelope stress response that promotes redistribution of cardiolipin microdomains. The second mechanism occurs through electrostatic repulsion by increasing the net positive charges at the cell surface, preventing the binding of the calcium-daptomycin complex (Fig. 8B). In S. aureus this occurs through the incorporation of positively charged lysine residues into the peptidoglycan through MprF.

Another repulsion mechanism relies on the incorporation of the positively charged amino acid D-alanine into cell wall teichoic acids. This mechanism is encoded by the dlt operon in S. aureus and E. faecium (Tran et al. 2015, Heidary et al. 2018). Lastly, a cardiolipin synthase has also been described to be involved in the repulsion mechanism in S. aureus and E. faecium (Patel et al. 2006) (Table 2).

Figure 8. The two proposed mechanisms of daptomycin resistance. (A) In the first one, cardiolipins are redistributed away from the septum (black arrow), which results in diversion of daptomycin from the binding site and thereby, prevents daptomycin binding. (B) The second mechanism involves electrostatic repulsion of the calcium-daptomycin complex from the cell membrane upon increase of the net positive charges of the cell envelope. Adapted from (Heidary et al. 2018).

3. Antimicrobial peptides with broad spectrum activity 3.1 Host defense peptides

Host defense peptides or innate immunity peptides can be found in nature as part of the immune defense of many living organisms (Wang and Wang 2004, Jenssen, Hamill and Hancock 2006, Zasloff 2002). Host defense peptides able to kill bacteria like the cecropins isolated from Hyalophora cecropia (Saugar et al. 2006, López-Rojas et al. 2011), the human LL-37 (Wang et al. 2014), and defensins from mammalian and invertebrate hosts have become a main focus for the development of new drugs (Ganz et al. 1985, Verma et al. 2007).

More than 2000 AMPs have been discovered with eukaryotic origin (Wang and Wang 2004, da Costa et al. 2015) and even though they have comparable antimicrobial activity, they are

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