AmpC-inducible expression

P. aeruginosa possesses an inducible chromosomally-encoded AmpC cephalosporinase which is similar to that found in several members of the Enterobacteriaceae [Jacoby GA, 2009]. According to the Bush-Jacoby-Medeiros classification, AmpC is a serine β-lactamase belonging to group I and, based on the Ambler structural classification, to class C β-lactamases. Possibly, AmpC is the most relevant antibiotic resistance mechanism of this opportunistic pathogen.

WT P. aeruginosa strains produce only low basal amounts of this enzyme remaining susceptible to antipseudomonal penicillins, penicillin-inhibitor combinations, antipseudomonal cephalosporins (ceftazidime and cefepime) and carbapenems.

Nevertheless, AmpC production can significantly be increased under particular circumstances, conferring resistance to all β-lactams. AmpC increased production can occur either, through mutations within its regulatory genes (section 1.8.) or by induction of the ampC gene. AmpC induction is a reversible process which occurs under exposure tospecific β-lactams and β-lactamase inhibitors such as cefoxitin, imipenem and/or clavulanate [Lister PD et al, 2009]. As following detailed, AmpC induction is a complex process intimately linked with peptidoglycan (PGN) recycling (Figure 1.1.).

The PGN of P. aeruginosa is built up of chains with n repeats of the disaccharide monomer N-acetyl-glucosamine-N-acetyl-muramic-acid (GlcNAc-MurNAc) connected to other identical chains by stem peptides linked to the MurNac units. The stem peptide from a disaccharide monomer is originally a pentapeptide (L-Alanine-D-Glutamicacid-diaminopimelicacid-D-Alanine-D-Alanine) and connects to a second stem peptide from another disaccharide monomer located on a different chain thanks to the transpeptidase activity of the high molecular mass penicillin-binding proteins (PBP1, PBP2 and PBP3). These PBPs cleave the terminal D-Alanine from the first pentapeptide (carboxypeptidase activity), converting it into a tetrapeptide which eventually binds to the diaminopimelic acid from other pentapeptide (transpeptidation). Thus, these bonds allow for the crosslinking of disaccharide chains which constitute the essential PGN architecture. Once the basic PGN structure is built, some other PBPs, mainly the low molecular mass PBPs (PBP4, PBP5 and PBP6) are thought to finely shape it. These PBPs exert D-carboxypeptidase activities and are known to release the terminal D-Ala from pentapeptides not destined to be cross-linked converting them into tetrapeptides not suitable for transpeptidation [Juan C et al, 2017].

Figure 1.1. Schematic representation of the interplay between PGN recycling, ampC regulation (induction) and intrinsic β-lactam resistance in P. aeruginosa. From: Juan C et al, 2017.

On each generation P. aeruginosa naturally degrades about 50% of its PGN mainly thanks to the action of the periplasmic autolysins (endopeptidases), which break the abovementioned bonds originating not cross-linked peptides, and to the action of the lytic transglycosylases, which break the bonds between the disaccharide units. Up to 90% of the degraded PGN is thought to be recycled, which supposes an outstanding resource-saving strategy. The action of the cited periplasmic enzymes results mainly in

GlcNAc-1,6-anhydro-MurNAc tri-, tetra- and penta- peptides [Vollmer W & Höltje JV, 2001], resulting fragments that are transported through the permease AmpG into the cytosol [Korfmann G & Sanders CC, 1989; Dietz H & Wiedemann B, 1996; Cheng Q & Park JT, 2002]. This is a key step for the downstream AmpC regulation and recycling events, as AmpG is the specific door for the entrance of PGN-derived mediators with AmpC regulator capacity [Zamorano L, 2011]. Once in the cytosol, the cytosolic L, D-carboxypeptidase LdcA cleaves the D-Ala from the tetrapeptides units, avoiding the potential accumulation of UDP-MurNAc tetrapeptides which are thought to be toxic for the bacterial cell [Templin MF et al, 1999]. As well, a glycoside hydrolase called NagZ removes the GlcNAc residues [Zamorano L et al, 2010] resulting in a pool of cytosolic GlcNAc units plus 1,6-anhydro-MurNAc peptides [Cheng Q et al, 2000;

Vötsch W & Templin MF, 2000] that, in non-inducer standard conditions, would eventually be recycled into UDP-MurNAc pentapeptides and exported to the nascent PGN.

Classically, it has been believed that the 1,6-anhydro-MurNAc tri- and penta- peptides units [Jacobs C et al, 1994; Dietz H et al, 1997] are signal molecules that induce ampC transcription and, indeed, the UDP-MurNAc pentapeptide has been identified as a repressor of ampC transcription to basal levels. Thus, these metabolites have been suggested to competitively regulate ampC transcription by directly binding to the LysR-type transcriptional regulator AmpR [Jacobs C et al, 1994]. AmpR and AmpC coding genes are located next to each other within the genome, divergently codified and with overlapping promoter regions to which AmpR binds to regulate their transcription [Lindquist S et al, 1989; Bartowsky E &

Normark S, 1993]. Under non-inducer standard conditions, the cytosolic AmpD, through its N-acetyl-muramyl-L-alanine amidase activity, cleaves the stem peptide from both the GlcNAc-1,6-anhydro-MurNAc and the 1,6-anhydro-MurNAc peptides [Höltje JV & Glauner B, 1990; Jacobs C et al, 1994], which results in low amounts of activation ligands. On the contrary, the amount of UDP-MurNAc pentapeptides can be increased thanks to the anabolic pathways starting from the pool of AmpD cleaved peptides and, thus, can both, enter into the PGN recycling route and bind to AmpR promoting the formation of an AmpR- deoxyribonucleic acid (DNA) complex that represses ampC transcription to basal levels.

In this sense, it has been proposed that exposure to certain β-lactams known to be AmpC inducers, such as cefoxitin and imipenem, triggers the accumulation of 1,6-anhydro-MurNAc peptides within the cytosol, reaching levels that cannot be efficiently processed by AmpD [Dietz H & Wiedemann B, 1996; Wiedemann B et al, 1998; Vollmer W & Höltje JV, 2001].

This accumulation would presumably displace the UDP-MurNAc pentapeptide from AmpR, generating a new complex that would act as an activator of ampC transcription and, thus, leading to clinically significant resistance against the inducer and other hydrolysable β-lactams [Jacobs C et al, 1994]. The molecular basis for the mentioned increase in the 1,6- anhydro-MurNAc pentapeptides amount during induction is believed to be related with the capacity of the inducer β-lactams to inhibit the DD-carboxypeptidase activity of the low

molecular mass PBPs [Sanders CC et al, 1997; Tayler AE et al, 2010; Fisher JF &

Mobashery S, 2014]. In this sense, Moyà et al. showed that the inducer β-lactams can inhibit the non-essential low molecular mass PBP4 (dacB) [Moyà B et al, 2009], affecting the PGN composition and favouring the entrance of activation ligands through AmpG. Interestingly, the authors also showed that PBP4 inducer-inhibition additionally triggers the activation of the two-component system CreBC which plays a collateral and minor role during the process. Thus, it has been proposed that PBP4 acts as a sentinel for the cell wall damage caused by the inducers, triggering an AmpR-dependent overproduction of AmpC and activating the CreBC system. The induction mechanism is a reversible process and ampC expression returns to basal levels in the absence of the antibiotic inducers [Mark BL et al, 2012]. Also it should be highlighted that the hydrolytic effect of AmpC onto a β-lactam will not only depend on the antibiotic inducer capacity but also on the hydrolysing efficiency of the cephalosporinase. Therefore, the inducible expression of AmpC plays a major role in the intrinsic resistance of P. aeruginosa to aminopenicillins and most cephalosporins (particularly cephamycins such as cefoxitin) since these molecules are potent inducers of the expression and efficiently hydrolyzed by this enzyme. Likewise, the inducible AmpC plays a major role in the basal reduced susceptibility level of P. aeruginosa to the carbapenem imipenem, as the relatively stability of this molecule to the hydrolysis by the cephalosporinase is to some extent compromised by its extremely high potency as inducer [Livermore DM, 1992].

Non-reversible mutational derepression leading to constitutive high-level expression of AmpC will be discussed later in section 1.8.