This work establishes point mutations in pmrB
as a regulatory mechanism of Pm resistance in CF isolates of P. aeruginosa
. The pmrB
mutants were cultured from CF patients at clinical centers in Denmark and the United Kingdom, where inhaled CST is often used as a long-term treatment for chronic airway infection (18
). Epidemic spread of Pmr P. aeruginosa
occurred in Copenhagen, Denmark, and Leeds, United Kingdom (14
), representing a considerable concern from an infection control standpoint. However, the pmrB
mutants in Copenhagen apparently arose sporadically and did not spread from patient to patient; the Copenhagen strain that did spread extensively has a resistance-conferring phoQ
). Four of the pmrB
mutants from Denmark also have mutant phoPQ
alleles: PA1603 has phoP24Q24
, PA1571 has phoQ25
, and PA1016 and PA1017 have phoQ30
. However, these native phoPQ
alleles appear not to confer Pm resistance (39
All three Pmr clinical strains in Leeds are pmrB mutants; however, only one of the Leeds pmrB alleles (pmrB22) confers moderate-level Pm resistance. The isolate that carries this allele (PA1133) also has a mutant phoQ allele that appears not to confer Pm resistance (S. M. Moskowitz, M. K. Brannon, and A. K. Miller, unpublished results). The Leeds strain that spread from patient to patient (represented here by isolate PA1131) has a mutant pmrB allele (pmrB42) that does not confer Pm resistance or induce arnB transcription in a ΔpmrAB background. It also has a mutant phoPQ allele that appears not to confer Pm resistance (Moskowitz et al., unpublished results); thus, the resistance-conferring genetic determinant(s) in this strain has not been defined.
In contrast to Salmonella enterica
, in which a prototypical resistance-conferring mutation occurred in the cognate response regulator PmrA (50
), the few PmrA mutations that we have found in P. aeruginosa
, exemplified by the pmrA2
allele in isolate PA1131, neither suppress nor augment Pm resistance in a pmrB
“gain-of-resistance” mutant background. We have also sequenced the promoter region of the arnB
operon in several highly Pmr
clinical isolates without defining any mutations that could plausibly contribute to Pm resistance (S. M. Moskowitz and M. K. Brannon, unpublished results).
Mutations in pmrB
similar or identical to some of those described here have been reported for non-CF clinical isolates of P. aeruginosa
with low- to moderate-level Pm resistance. For example, the recombinant allele that we have designated pmrB32
contains a mutation that changes Met 292 to Ile and confers low-level Pm resistance when expressed in a ΔpmrAB
strain background; the same residue was mutated to Thr in a non-CF clinical isolate from New York with a PMB MIC of 8 mg/liter (1
). The mutant allele designated pmrB34
contains a mutation that changes Ala 248 to Thr and confers low-level Pm resistance; the same residue was mutated to Val in a laboratory mutant with moderate Pm resistance (42
), and the adjacent residue (Ala 247) was mutated to Thr in a different non-CF isolate from New York with a PMB MIC of 8 mg/liter (5
). The mutant allele designated pmrB45
, which was found in a moderately Pmr
CF isolate from the United Kingdom, also occurred in a Brazilian non-CF clinical isolate with a CST MIC of 32 mg/liter (51
). These data suggest that mutations in specific residues of PmrB, particularly in the DHP domain and its catalytic H box, can represent first-step events that confer Pm resistance in P. aeruginosa
Several mutant pmrB alleles found in highly Pmr CF isolates had two nonsilent mutations, one in the periplasmic domain and another in the DHP domain. These double mutant alleles conferred moderate Pm resistance (CST MIC of 128 to 256 mg/liter) in a ΔpmrAB background. Recombinant pmrB alleles with only one of the two mutations conferred much less Pm resistance, in most cases only 1 to 2 dilutions above that conferred by a WT allele, indicating that periplasmic and DHP missense mutations have synergistic effects resulting in marked activation of the PmrAB system.
Interestingly, recombinant double mutant pmrB alleles (i.e., novel pairs of periplasmic and DHP mutations) conferred moderate levels of Pm resistance similar to the levels observed for the original double mutant alleles. This suggests that any of the specific point mutations observed in the periplasmic domain can act as a second-step event in a DHP domain mutant, altering the periplasmic domain conformation such that kinase activity is enhanced and/or phosphatase activity is inhibited in the altered DHP domain. For example, in the pmrB34 allele, an Ala 248-to-Thr mutation in the DHP domain appears as a first-step PmrB mutation. In the pmrB26 and pmrB41 alleles, this DHP domain mutation is combined with a periplasmic domain mutation (Ala 54 to Val and Arg 57 to His, respectively), representing a second mutational step.
The allelic expression plasmids conferred Pm resistance that was 2 to 32 times higher in a ΔpmrAB
strain background than in a WT background. Similarly, Abraham and Kwon observed that episomal expression of WT pmrAB
suppresses the Pm resistance of the pmrB
M292T allele (1
). These observations are consistent with the hypothesis that expression of a WT pmrAB
allele can partially or completely suppress the phenotypic effects of a mutant allele, regardless of whether the expression of either is chromosomal or episomal. This is probably a functional consequence of PmrB dimerization: whereas mutant-mutant homodimers likely lack PmrB phosphatase activity, the mutant-WT heterodimers formed when both alleles are expressed (49
) would be expected to preserve some PmrB phosphatase activity and thus limit the degree of Pm resistance conferred.
However, even in a ΔpmrAB
strain background, the Pm resistance of pmrAB
allelic expression strains was less than that of the corresponding Pmr
clinical isolates in which the mutant pmrAB
alleles were identified. One possible explanation is that the PmrAB system is autoregulated (i.e., is subject to transcriptional positive feedback) under the control of its native promoter (as in the clinical isolates); this positive feedback may be enhanced further by induction of mutant PmrB in the presence of Pm (38
). Mutations in additional loci, such as genes that influence the structure of the lipid A and core oligosaccharide moieties of LPS, may further augment Pm resistance in the clinical isolates.
A wide variety of structural modifications can be seen in lipid A from CF isolates of P. aeruginosa
), and most, if not all, of these variations were observed among the highly Pmr
isolates that were studied here. The patterns of lipid A modification among the Pmr
isolates did not correlate with specific pmrB
alleles. However, for unpassaged pmrB
mutants and their corresponding Pms
controls, we observed an absolute correlation between l
-Ara4N addition to lipid A and clinical Pm resistance. Upon passage, some of the pmrB
mutants lost Pm resistance but maintained l
-Ara4N addition. Taken together, these observations suggest that l
-Ara4N addition is necessary but not sufficient for Pm resistance. As previously observed in a preliminary analysis (40
), loss of lauroyl 2-hydroxylation also correlated strongly with clinical Pm resistance. It is not known whether the dephosphorylated minor species frequently observed in lipid A analyses actually contribute to Pm resistance or are merely a technical artifact of lipid A preparation.
In addition to demonstrating variations in lipid A structure, we showed that some clinical pmrB
mutants of P. aeruginosa
have a relatively stable Pm resistance phenotype even after 15 days of passage without antibiotic selection pressure, while others may completely lose resistance within 5 to 10 days. Thus, in some instances, pmrB
mutation appears to impose fitness costs, such that mutants are outcompeted by revertants (or WT strains) when both are present in an ecological niche or in vitro
culture without Pm selection pressure. Such phenotypic instability has also been observed for some clinical phoQ
mutants (S. M. Moskowitz and M. Pier, unpublished results). This loss of resistance suggests the emergence of secondary suppressor mutations. In clinical phoQ
mutations may act as partial or complete secondary suppressors of Pm resistance (39
). We also observed phenotypic instability while analyzing Pmr
strains through targeted gene deletions, which are typically constructed in the absence of Pm selection pressure. Loss of Pm resistance in a targeted gene deletion mutant that episomal expression fails to complement can sometimes be attributable to emergence of secondary suppressor mutations during strain construction. As a consequence, we have adopted the practice of first constructing targeted gene deletions in a WT strain background and then introducing the Pm resistance-conferring allele or mutation via deletion or allelic replacement as the last step prior to strain testing.
Abraham and Kwon showed that episomal expression of arnBCADTEF
completely complements the genetic disruption of arnB
in a mildly Pmr
). In contrast, although we were able to show through construction of clean deletions in CF isolate PA1016 that its Pm resistance is dependent on pmrAB
, episomal expression of the native alleles (pmrAB23
, respectively) only partially restored Pm resistance. Similar partial complementation was also observed in a WT strain background. This implies that inducible episomal expression of ArnC is not as robust as its native chromosomal expression, limiting Pm resistance under the conditions used here to assess this phenotype.
In summary, alterations in the PmrAB system caused by specific mutations in the pmrB gene represent an important but nonexclusive regulatory mechanism of clinical Pm resistance. Such mutations can cause low to moderate Pm resistance but also contribute to high-level resistance. Specific residues in the periplasmic and DHP domains of PmrB are mutated repeatedly in Pmr strains and appear to act synergistically to confer markedly increased resistance on periplasmic-DHP domain double mutants. Both single and double mutant pmrB alleles drive expression of the arnB operon, resulting in l-Ara4N addition to lipid A as an important biochemical mechanism of Pm resistance. Additional changes in LPS, such as loss of lauroyl 2-hydroxylation in lipid A, are associated with high-level Pm resistance, though a causative role is not yet established. Conversely, some highly Pmr isolates gradually lose resistance when passaged repeatedly without antibiotic selection pressure. Such loss of resistance does not consistently correlate with loss of l-Ara4N addition, because some secondary suppressor mutations evidently interfere with resistance in ways that leave this lipid A modification intact. Defining such secondary suppressors is likely to provide new insights into the regulatory and biochemical mechanisms that contribute to high-level Pm resistance in P. aeruginosa.