Identification of Two Late Acyltransferase Genes Responsible for Lipid A Biosynthesis in Moraxella catarrhalis

Abstract

Lipid A is a biological component of the lipooligosaccharide (LOS) of a human pathogen, Moraxella catarrhalis. No other acyltransferases except UDP-GlcNAc acyltransferase responsible for lipid A biosynthesis in M. catarrhalis have been elucidated. By informatics, two late acyltransferase genes lpxX and lpxL responsible for lipid A biosynthesis were identified, and knockout mutants of each gene in M. catarrhalis strain O35E was constructed and named as O35ElpxX and O35ElpxL. Structural analysis of lipid A from the parental strain and derived mutants showed that O35ElpxX lacked two decanoic acids (C10:0) while O35ElpxL lacked one dodecanoic (lauric) acid (C12:0), suggesting that lpxX gene encoded decanoyl transferase and lpxL gene encoded dodecanoyl transferase. Phenotypic analysis revealed that both mutants were similar to the parental strain in their toxicity in vitro. However, the O35ElpxX was sensitive to bactericidal activity of normal human serum and hydrophobic reagents. It had a reduced growth rate in broth and an accelerated bacterial clearance at 3 h (p <0.01) or 6 h (p <0.05) after an aerosol challenge in a murine model of bacterial pulmonary clearance. Meanwhile, the O35ElpxL presented alike patterns as the parental strain except it was slightly sensitive to the hydrophobic reagents. These results indicate that these two genes, particularly lpxX, encoding late acyltransferases responsible for incorporation of the acyloxyacyl linked secondary acyl chains into the lipid A are important for biological activities of M. catarrhalis.

Introduction

Moraxella catarrhalis is the third most common isolate following Streptococcus pneumoniae and nontypeable Haemophilus influenzae as the causative agent of otitis media in infants and young children [1–3]. In developed countries, more than 80% of children under the age of 3 years will be diagnosed at least once with otitis media, and M. catarrhalis is responsible for 15 to 25% of all of these cases [4, 5]. In adults with chronic obstructive pulmonary disease (COPD), which is the fourth leading cause of death in the United States, this organism is known to be the second cause of exacerbations of lower respiratory tract infections [6, 7]. Approximately 20 million cases of such exacerbations are reported each year in the United States, up to 35% of them resulting from M. catarrhalis infections [8]. In immunocompromised hosts, M. catarrhalis causes a variety of severe infections including septicemia and meningitis. Clinical and epidemiological studies revealed high carriage rates in young children and suggested that a high rate of colonization was associated with an increased risk of the development of M. catarrhalis-mediated diseases [3]. Currently, the molecular pathogenesis of M. catarrhalis infection is not fully understood.

As a Gram-negative bacterium without capsular polysaccharides, M. catarrhalis is surrounded by an outer membrane consisting of lipooligosaccharide (LOS), outer membrane proteins and pili outside phospholipids [3]. LOS is a major outer membrane component of M. catarrhalis with three major LOS serotypes, A, B and C [9–12]. Quite a few studies have demonstrated that LOS is an important virulence factor for many respiratory pathogens, such as Neisseria meningitidis and Haemophilus influenzae [13–15]. Studies have also implicated that M. catarrhalis LOS is important in the pathogenesis of M. catarrhalis infection [16–19]. In contrast to the LOS or LPS molecules from most Gram-negative bacteria, M. catarrhalis LOS consists of only an oligosaccharide (OS) core and lipid A [9]. The inner core OS is attached to 3-deoxy-D-manno-octulosonic acid (Kdo) through a glucosyl residue instead of a heptosyl residue [10, 20] while the lipid A portion consists of seven shorter fatty acid residues (decanoyl or dodecanoyl, C10:0 or C12:0) [10, 11].

Recently, several genes associated with LOS biosynthesis of M. catarrhalis, especially for the core OS moiety, were reported. Zaleski et al. identified a galE gene encoding UDP-glucose-4-epimerase in M. catarrhalis and showed inactivation of the gene resulting in an LOS lacking two terminal galactosyl residues [21]. Luke et al. showed a kdsA gene encoding Kdo-8-phosphate synthase and found a kdsA mutant consisting only of lipid A on its LOS molecule [22] while Peng et al. identified a kdtA gene encoding Kdo transferase during the LOS biosynthesis (18). Edwards et al. revealed a cluster of three LOS glycosyltransferase genes (lgt) for extension of OS chains to the inner core [23] and an lgt4 in serotype A and C strains [24]. Subsequently Wilson et al. found the lgt5 gene encoding an α-galactosyltransferase for addition of the terminal galactose of the LOS [25] while Schwingel et al. found the lgt6 gene involved in the initial assembly of the LOS [26]. However, as for the lipid A biosynthesis of the M. catarrhalis LOS, only an lpxA gene encoding UDP-N-acetylglucosamine acyltransferase responsible for the first step of lipid A or LOS biosynthesis in M. catarrhalis was identified and characterized [19]. Little is known regarding the late steps of the lipid A biosynthesis, particularly in the addition of the decanoyl and dodecanoyl acyloxyacyl residues.

Our knowledge of the enzymology and molecular genetics of the lipid A biosynthesis is based mainly on the studies of the LPS expressed by the enteric bacterium, especially Escherichia coli. The last steps of E. coli lipid A biosynthesis involve the addition of lauroyl and myristoyl residues to the distal glucosamine unit, generating acyloxyacyl moieties. The E. coli lauroyl and the myristoyl transferases are encoded by lpxL and lpxM, respectively, known as htrB and msbB prior to elucidation of their functions [20]. In this study, we identified two late acyltransferase genes encoding decanoyl transferase and dodecanoyl transferase from M. catarrhalis serotype A strain O35E and constructed the corresponding isogenic mutants. Analysis of physiochemical and biological features of both mutants was performed to study the functions of these genes and the structures of their resultant LOSs in vitro and in vivo.

Results

Identification of putative late acyltransferase genes of M. catarrhalis O35E

Two putative late acyltransferase genes in M. catarrhalis strain O35E were identified by BLAST searching from the M. catarrhalis partial genome sequence (AX067448 and AX067465). According to the sequence analysis results and structural data of each lipid A, these two genes were named as lpxX and lpxL. Analysis of promoter and ORF showed that the lpxX or lpxL DNA fragment contained a single ORF of 924 or 978 bp with a predicted gene product of 307 or 325 amino acids (Fig. 1). Upstream sequence analysis of the lpxX gene revealed the presence of a gene encoding aspartyl-tRNA synthetase (ats), while sequence analysis for the downstream region of the lpxX revealed the presence of a glycosyltransferase (lgt 6) gene (Fig. 1A) [26]. The upstream gene of lpxL was atr (encoding ABC transporter related protein), and the downstream gene was asd (encoding aspartate 1-decarboxylase) (Fig. 1B). The deduced amino acid sequences of lpxX and lpxL showed 19–32% identity and 39–50% similarity compared with the identified late acyltransferase homologues of other Gram-negative bacteria (Table 2). However, the identity and similarity between lpxX and lpxL were only 22% and 37%, respectively. Protein sequence analysis of M. catarrhalis LpxX and LpxL revealed that both contained membrane-spanning regions anchoring the proteins to the inner membrane but not in the cytoplasm of the bacterium (Data not shown), which is consistent to those of defined E. coli late acyltransferases [27]. The transmembrane helix locations and topology structures of LpxX and LpxL were also similar with that of E. coli late acyltransferases.

Fig.1.

Genetic organization of lpxX or lpxL locus in M. catarrhalis O35E genome. (A) The location of deletion in lpxX replaced by a zeocin resistance (Zeo^r) gene is between two EcoRI cleavage sites introduced by PCR reaction. A gene upstream from lpxX is an aspartyl-tRNA synthetase (ats) while a downstream gene encodes a glycosyltransferase (lgt 6). (B) The location of deletion in lpxL replaced by a kanamycin resistance (Kan^r) gene is between two PstI cleavage sites. A gene upstream from lpxL is an ABC transporter related (atr) while a downstream gene encodes an aspartate1-decarboxylase (asd). Large arrows represent the direction of transcription, and the sites of primers used are indicated with small arrows.

TABLE 2.

Comparison of M. catarrhalis LpxX and LpxL with the late acyltransferase homologues identified in other Gram-negative bacteria

Bacterium	Protein (Accession no.)	Identity	Similarity	Reference
LpxX
Escherichia coli str. K12	LpxL (NP_415572)	24% (68/281)	41% (117/281)	27
substr. W3110	LpxP (NP_416879)	21% (53/243)	44% (109/243)	28
Neisseria meningitidis	MsbB (AAL74160)	24% (70/281)	43% (121/281)	29
Neisseria gonorrhoeae	MsbB (AAL24441)	24% (68/278)	43% (122/278)	30
Haemophilus influenzae	HtrB (P45239)	23% (66/283)	41% (118/283)	31
Rd KW20
Salmonella typhimurium	HtrB (NP_460126)	23% (64/273)	39% (108/273)	32
LT2
Francisella tularensis	HtrB (YP_666416)	22% (57/253)	43% (110/253)	33
subsp. Tularensis
Yersinia pestis KIM	MsbB (AAM85807)	19% (54/272)	39% (105/272)	34
LpxL
Escherichia coli str. K12	LpxL (NP_415572)	31% (96/305)	49% (150/305)	27
substr. W3110	LpxP (NP_416879)	31% (96/306)	50% (156/306)	28
	LpxM (AP_002475)	27% (87/319)	46% (147/319)	35
Neisseria meningitidis	MsbB (AAL74160)	26% (67/252)	44% (111/252)	29
Neisseria gonorrhoeae	MsbB (AAL24441)	27% (74/270)	44% (121/270)	30
Haemophilus influenzae	HtrB (P45239)	32% (101/308)	50% (156/308)	31
Rd KW20	MsbB (NP_438368)	28% (90/316)	48% (154/316)
Salmonella typhimurium	HtrB (NP_460126)	31% (96/304)	48% (146/304)	32
LT2	MsbB (AAL20805)	26% (85/319)	46% (147/319)
Francisella tularensis	HtrB (YP_666416)	26% (81/309)	45% (142/309)	33
subsp. Tularensis
Yersinia pestis KIM	MsbB (AAM85807)	27% (88/321)	46% (149/321)	34

Construction and characterization of lpxX and lpxL knockout mutants

The lpxX mutant was constructed by allelic exchange of a 53-bp deletion within the induced EcoRI sites of the lpxX coding region with a substitution of a zeocin-resistant (Zeo^r) cassette, and the lpxL knockout mutant was constructed by allelic exchange of a 454 bp deletion between two PstI sites of the lpxL coding region with a substitution of a kanamycin-resistant (Kan^r) cassette (Fig. 1). Nucleotide sequence analysis of PCR products confirmed that the cassettes had been inserted into the chromosomal DNA at the predicted positions. The mutant bacteria were named as O35ElpxX and O35ElpxL.

Southern blot was performed to test if a single copy of the Zeo^r or Kan^r gene was inserted into the genome, the O35ElpxX or O35ElpxL genomic DNA was digested with EcoRV (Fig. 2A/C) and probed with DIG-labeled Zeo^r gene or DIG-labeled Kan^r gene, respectively (Fig. 2B/D). Only one band was detected in the chromosomal DNA of the O35ElpxX or O35ElpxL (Fig. 2B, lane 2; Fig. 2D, lane 4), but none in that of the parental O35E (Fig. 2B/D, lane 1), showing a single insertion in the genome of each mutant.

Fig.2.

Detection of Zeo^r gene inserted into O35ElpxX or Kan^r gene into O35ElpxL chromosomal DNA by Southern blotting. Lane 1, 2 or 4, 5 μg of chromosomal DNA from O35E, O35ElpxX or O35ElpxL plus EcoRV; lane 3, 0.1 μg of pEM7/Zeo (a plasmid with a Zeo^r cassette as positive control) plus EcoRI+XhoI; lane 5, 0.1 μg of pUC4K (a plasmid with a Kan^r cassette as positive control) plus EcoRI. Each digested sample was resolved on a 0.7% agarose gel and visualized by ethidium bromide staining (A, C). Southern blotting was performed using DIG-labeled Zeo^r (B) or Kan^r gene probe (D). Lamda DNA/EcoRI-plus-HindIII molecular size standards (Fermentas) are shown in base pairs on the left (lane M).

To determine if the insertion had a polar effect on the upstream or downstream gene, total RNA isolated from the parental O35E, O35ElpxX or O35ElpxL was subjected to RT-PCR analysis using primer sets designed for genes ats [ats1/ats2], lpxX [b1SP/b1AP] and lgt 6 [lg1/lg2]; or genes atr [atr1/atr2], lpxL [b2SP/b2AP] and asd [asd1/asd2], respectively. When compared to the parental O35E, the insertion of the Zeo^r gene in the O35ElpxX only disrupted the lpxX gene transcription (Fig. 3A, lane 4b), and the insertion of the Kan^r gene in the O35ElpxL only disrupted the lpxL gene transcription (Fig. 3B, lane 7e).

Fig.3.

Detection of lpxX (A) and lpxL (B) gene expression by RT-PCR. The RT-PCRs were performed using the following nucleic acid templates and primers: total RNA from O35E (lanes 1 and 3), O35ElpxX (lanes 4 and 6) and O35ElpxL (lanes 7 and 9), and chromosomal DNA from O35E (lane 2), O35ElpxX (lane 5) and O35ElpxL (lane 8). Reaction sets contained the following primers: for lanes labeled “a”, ats1/ats2; lanes labeled “b”, b1SP/b1AP; lanes labeled “c”, lg1/lg2; lanes labeled “d”, atr1/atr2; lanes labeled “e”, b2SP/b2AP; and lanes labeled “f”, asd1/asd2. The controls (lanes 3, 6, 9) used total RNA as the nucleic acid template without activation of the RT. GenRuler DNA ladder mix (Fermentas) was used for the molecular size standards in base pairs (M).

Determination of LOSs in lpxX and lpxL mutants

An attempt was made to isolate LOS from proteinase K-treated cell lysates of O35E, O35ElpxX and O35ElpxL. Silver staining analysis after SDS-PAGE with three extracts revealed a different migration manner for the mutant LOS as compared to that of the parental LOS. In particular, for the O35ElpxX mutant LOS, the band was located below the O35E band (Fig. 4, lane 2), and had a reduced intensity and a conversion from a black to a brown coloration while the LOS migration of the O35ElpxL (Fig. 4, lane 4) was slightly below that of the parental LOS. After complementation of the parental lpxX or lpxL gene by pWlpxX or pWlpxL (Table 1), silver staining analysis with the revertant O35ElpxX or O35ElpxL strain showed that an LOS band migrated in a manner identical to that of the parental LOS. The LOS band of the revertant O35ElpxX strain also had a conversion from a brown to a black coloration (Fig. 4, lanes 3, 5).

Fig.4.

LOS patterns from SDS-PAGE followed by sliver staining. M. catarrhalis wild type strain O35E (lane 1), O35ElpxX, O35ElpxX revertant (lanes 2, 3), O35ElpxL and O35ElpxL revertant (lanes 4, 5). Extracts from proteinase K-treated whole-cell lysates from each bacterial suspension (1.9 μg of protein content) were used and molecular mass markers (Mark12; Invitrogen) indicated on the left.

TABLE 1.

Strains, plasmids and primers used in this study

	Description	Source
Strain
M. catarrhalis	Wild-type strain	46
O35E
M. catarrhalis	Decanoyl transferase-deficient strain	This study
O35ElpxX
M. catarrhalis	Dodecanoyl transferase-deficient strain	This study
O35ElpxL
E. Coli TOP10	Cloning strain	Invitrogen
Plasmid
pCR2.1	TOPO TA cloning vector	Invitrogen
pCRlpxX	lpxX cloned into pCR2.1	This study
pCRlpxL	lpxL cloned into pCR2.1	This study
pBluescript II	Cloning vector	Fermentas
SK(+)
pSlpxX	XhoI-BamHI lpxX fragment cloned into SK(+)	This study
pSlpxL	EcoRI-BamHI lpxL fragment cloned into SK(+)	This study
pEM7/Zeo	Zeocin-resistant cassette	Invitrogen
pSlpxX-zeo	Zeocin-resistant gene inserted into pSlpxX	This study
pUC4K	Kanamycin-resistance cassette	Amersham
pSlpxL-kan	Kanamycin-resistance gene inserted into pSlpxL	This study
pWW115	Cloning vector for use with M. catarrhalis	47
pWlpxX	BamHI-SacI lpxX fragment cloned into pWW115	This study
pWlpxL	BamHI-SacI lpxL fragment cloned into pWW115	This study
Primer
B1SP	5′-AGC TCA TCA GTG CAG TCG-3′(lpxX sense)	This study
B1AP	5′-CTT TGA CAT GGC TTG AAG-3′ (lpxX antisense)	This study
B1X	5′-CTC CTC GAG AGC TCA TCA GTG CAG TCG-3′ (lpxX sense; XhoI site underlined)	This study
b1B	5′-CTC GGA TCC CTT TGA CAT GGC TTG AAG-3′ (lpxX antisense; BamHI site underlined)	This study
b1E1	5′-CTC GAA TTC GTA TCA TAC TGC CCG ACC-3′ (Inserting zeo gene; EcoRI site underlined)	This study
b1E2	5′-CTC GAA TTC GTG GGT ACA AGG CTG GCA-3′ (Inserting zeo gene; EcoRI site underlined)	This study
b1B1	5′-CTC GGA TCC GTG CTT GGT TTT TTA AGA TAT GTA CC-3′ (lpxX sense; BamHI site underlined)	This study
b1S	5′-CTC GAG CTC TCA CTC ATA ACT ATC CTT TGA CAT GG-3′ (lpxX antisense; SacI site underlined)	This study
ats1	5′-GCT CAA TCC GTG ATG TGA-3′ (ats sense)	This study
ats2	5′-CGA CTG CAC TGA TGA GCT-3′ (ats antisense)	This study
lg1	5′-CTT CAA GCC ATG TCA AAG-3′ (lgt 6 sense)	This study
lg2	5′-CGA ATA ATC ATC ACA CTG-3′ (lgt 6 antisense)	This study
zeo EcoRI-1	5′-CTC GAA TTC CAC GTG TTG ACA ATT AAT-3′ (zeocin sense; EcoRI site underlined)	This study
zeo EcoRI-2	5′-CTC GAA TTC TCA GTC CTG CTC CTC GGC-3′ (zeocin antisense; EcoRI site underlined)	This study
b2SP	5′-GAG TTG CCA TCA TCA GCA-3′ (lpxL sense)	This study
b2AP	5′-AAT TGG TGT CAT CGG CTT-3′ (lpxL antisense)	This study
b2E	5′-CTC GAA TTC GAG TTG CCA TCA TCA GCA-3′ (lpxL sense; EcoRI site underlined)	This study
b2B	5′-CTC GGA TCC AAT TGG TGT CAT CGG CTT-3′ (lpxL antisense; BamHI site underlined)	This study
b2B1	5′-CTC GGA TCC TTG ACA GAT ACT CAT AAA CAA AGT AGC-3′ (lpxL sense; BamHI site underlined)	This study
b2S	5′-CTC GAG CTC TTA ATG TTG ATA GTA ATT GGT GTC A-3′ (lpxL antisense; SacI site underlined)	This study
atr1	5′-TGC TTG ATG AGC CTA CCA-3′ (atr sense)	This study
atr2	5′-TGC TGA TGA TGG CAA CTC-3′ (atr antisense)	This study
asd1	5′-AAG CCG ATG ACA CCA ATT-3′ (asd sense)	This study
asd2	5′-GCA GGT TCA TAG TGC ATG-3′ (asd antisense)	This study
Kan RP	5′-GGT GCG ACA ATC TAT CGA-3′ (kanamycin sense)	19
Kan FP	5′-CTC ATC GAG CAT CAA ATG-3′ (kanamycin antisense)	19

Composition and MALDI-TOF MS analysis of lipid A in lpxX and lpxL mutants

The fatty acid compositions of the lipid A liberated from strain O35E, O35ElpxX and O35ElpxL are shown in Fig. 5. The published lipid A structure of the M. catarrhalis serotype A strain ATCC 25238 is acylated with four molecules of 3OH-C12:0, two of C10:0 and one of C12:0 [10]. When compared to this structure, the lipid A of the O35ElpxX lacks two decanoic acyl (C10:0) substituents, and the O35ElpxL lacks one lauroyl acid (C12:0) substituents.

Fig.5.

GC-MS profiles of the fatty methyl esters (FAME) obtained from lipid A of M. catarrhalis wild type strain O35E, O35ElpxX and O35ElpxL. When compared with the lipid A of the parental strain O35E (A), the lipid A of O35E lpxX mutant did not contain C10:0 (decanoic acid) (B) while the O35ElpxL showed no C12:0 [dodecanoic (lauric) acid] (C).* indicates impurities.

MALDI-TOF MS analysis showed differences in the mass of lipid A from both mutants as compared to that of their parental strain (Fig. 6). The MS of the lipid A from strain O35E LOS (Fig. 6A) revealed the presence of three minor species of ions at m/z 1907.94, 1930.33, 1953.05 and a major ion at m/z 1784.75. The 1907.94 ion represented a lipid A that had the composition of published lipid A structure; i.e. P₂-PEA-GlcNAc₂-3OHC12:0₄-C10:0₂-C12:0₁ and its mono- and di-sodiated forms at m/z 1930.33 and 1953.05, respectively. The major observed ion at m/z 1784.75 is due to this structure that lacks a phosphoethanolamine (PEA) group (i.e. less 123 amu). The loss of the PEA group likely occurs due to the lability of its pyrophosphate bond, which can hydrolyze to the lipid A phosphate under mild acid hydrolysis conditions.

Fig.6.

MALDI-TOF analysis of the lipid A from M. catarrhalis wild type strain O35E, O35ElpxX and O35ElpxL, and their proposed structures. These analyses were done in the negative mode, and all ions were represented as deprotonated [M-H]⁻ ions. The upper portion of the figure shows the structure of the major species of O35E lipid A at 1907.94 amu (A). In contrast, the lipid A of the O35ElpxX was pentaacylated and lacked two C10:0 residues with a structure at 1599.25 amu (B), while the O35ElpxL lipid A was hexaacylated and missing one C12:0 residue with a structure at 1725.62 amu (C).

Compared to the lipid A of the parental LOS, the spectrum of lipid A from the O35ElpxX mutant (Fig. 6B) is consistent with a structure that lacks decanoic acid (C10:0). This result is consistent with data from fatty acid methyl esters (FAMEs) analysis (Fig. 5B). The lipid A from O35ElpxX revealed the presence of three major ions at m/z 1476.06, 1498.08, and 1520.17. These ions represented the structure P₂-GlcNAc₂-3OHC12:0₄-C12:0₁ lacked PEA group and its mono- and di-sodiated forms. The cluster of ions at m/z 1599.26, 1623.29 and 1646.28, respectively, represented the structure having the compositions: P₂-PEA-GlcNAc₂-3OHC12:0₄-C12:0₁ (1599.26) and its mono- and di-sodiated forms. The other ions at m/z 1395.97, 1417.86 and 1440.96 were due to a monophosphorylated structure P-GlcNAc₂-3OHC12:0₄-C12:0₁ (1395.97) and its mono- and di-sodiated forms. The ion at m/z 1293.62 was due to a monophosphorylated tetraacylated structure P-GlcNAc₂-3OHC12:0₄ and the ions at m/z 1315.67 and 1337.84 were mono- and di-sodiated forms, respectively.

Consistent with observation from FAMEs analysis for the lipid A from O35ElpxL which lacks lauric acid (C12:0), the MALDI-TOF spectrum (Fig. 6C) shows an ion at m/z 1725.62 which corresponds to a composition of P₂-PEA-GlcNAc₂-3OHC12:0₄-C10:0₂. Its mono- and di-sodiated forms are also present at m/z 1748.63 and 1770.65. The ion at m/z 1602.42 is due to a lipid A structure that lacks a PEA group (a loss of 123 mass units from m/z 1725.62) and corresponds to a composition of P₂-GlcNAc₂-3OHC12:0₄-C10:0₂. The ions at m/z 1625.44 and 1645.53 are mono- and di-sodiated species. The ion at m/z 1448.05 is due to a structure that has a composition of P₂-GlcNAc₂-3OHC12:0₄-C10:0₁, and the m/z 1470.08 and 1492.20 ions are its mono- and di-sodiated forms, respectively (Fig. 6C).

Composition and structural analysis of OSs in LOSs of lpxX and lpxL mutants

The OS portion of each LOS was obtained after mild acid hydrolysis and analyzed for its glycosyl compositions and by MALDI-TOF MS (Fig. 7). Glycosyl composition analyses of the OS from either O35ElpxX or O35ElpxL LOS all show a glycosyl residue ratio of Gal₂Glc₅GlcNAc₁Kdo, which is consistent with the glycosyl components of the published serotype A structure for the parental strain [10]. These results are consistent with the conclusion that the OS from O35ElpxX and O35ElpxL mutants have the same structure as that of the parental strain O35E.

Fig.7.

The MALDI-TOF MS spectra for the OSs released from the O35ElpxX (A) and O35ElpxL (B) mutants. The inset shows the compositions and the calculated ions for the observed ions in each of these spectra.

Morphology and growth rate of lpxX and lpxL mutants

The O35ElpxX formed small and transparent colonies on the chocolate agar plates when compared with the parental strain. When it grew in BHI broth, its growth rate was slower than the parental strain in logarithmic phase (Fig. 8A). The colonies of reverted O35ElpxX with pWlpxX (Table 1) were similar to those of the wild type strain (data not shown). For the O35ElpxL mutant, the colonies on the chocolate agar plates were similar to those of the parental strain, and the growth rate in BHI broth in logarithmic phase was also similar to that of the parental strain (Fig. 8A).

Fig.8.

Growth curves, bactericidal resistance and mouse clearance of M. catarrhalis wild type strain O35E, O35ElpxX and O35ElpxL. (A) Strain O35E (□), O35ElpxX (■) or O35ElpxL ( ) was grown in BHI broth at 37°C, respectively, and their optical density checked at different times. (B) Bactericidal activities of normal human serum against strain O35E (white bar), O35ElpxX (black bar) and O35ElpxL (gray bar) were shown. “HI” represents the group of 25% heat-inactivated normal human serum as controls for each strain tested. The data represent the averages of three independent assays. (C) Time courses of bacterial recovery in mouse lungs after an aerosol challenge with strain O35E (□), O35ElpxX (■) and O35ElpxL ( ). Each time point represents a geometric mean of eight mice. ^*: p<0.05; ^**: p<0.01

Susceptibility of lpxX and lpxL mutants

A broad range of hydrophobic agents and a hydrophilic glycopeptide were used to determine the susceptibility of both mutants. Both mutants, especially O35ElpxX, exhibited more susceptibility to most hydrophobic antibiotics and reagents than that of the parental strain, except that O35ElpxL was more resistant to deoxycholate than the parental strain. Both O35ElpxX and O35ElpxL showed a similar resistance to the hydrophilic glycopeptide, vancomycin, as the parental strain (Table 3).

TABLE 3.

Susceptibility of M. catarrhalis wild-type O35E and its lpxX and lpxL mutants to a panel of hydrophobic reagents or hydrophilic glycopeptide

Compound	Zone of growth inhibition (mm) for strain a
	O35E	O35ElpxX	O35ElpxL
Clindamycin (2μg)	11.5±0.5	14.5±0.5	11.5±0.8
Fusidic acid (10mg/ml)	22.2±0.3	28.8±0.3	24.9±0.7
Novobiocin (5μg)	13.8±0.3	16.7±0.6	14.7±0.3
Polymycin B (300iu)	12.0±0.2	14.0±0.3	13.8±0.3
Rifapin (5μg)	21.5±0.5	32.3±0.6	25.7±0.6
Vancomycin (5μg)	<6.0b	<6.0	<6.0
Deoxycholate (100mg/ml)	19.2±0.3	22.8±0.3	17.8±0.8
Triton X-100 (5%[wt/vol])	18.3±0.3	25.0±0.5	20.3±0.3
Tween 20 (5%[vol/vol])	15.8±0.3	21.2±0.8	18.2±0.8
Azithromycin (15μg)	22.3±0.6	34.2±0.8	29.4±0.5

^aSensitivities were assessed by measuring the diameters of the zones of growth inhibition on two axes, and the mean values were calculated. The data represent the averages of three separate experiment ± SD.

^bNo inhibition.

Biological activities of lpxX and lpxL mutants

The O35ElpxX and O35ElpxL mutants were tested for LOS–associated biological activity. In an LAL assay, whole cell suspensions (A₆₂₀ = 0.1) gave 2.24 × 10³ endotoxin units (EU)/ml for O35E, 7.26 × 10³ EU/ml for O35ElpxX and 6.05 × 10³ EU/ml for O35ElpxL, respectively.

In a bactericidal assay, 87.7% and 87.4% of cells from strain O35E survived at 12.5% and 25% of normal human serum, respectively (Fig. 8B). However, only 50.3% or 34.5% (p<0.05) of cells from the O35ElpxX mutant survived at 12.5% or 25% of normal human serum while no difference was found between the O35ElpxL mutant and the parental strain, indicating a reduced resistance to the normal human serum from the O35ElpxX.

In a murine respiratory clearance model after an aerosol challenge with each viable bacterium, the O35ElpxL showed a similar bacterial clearance pattern as the parental strain. However, the number of O35ElpxX cells presented in mouse lungs was approximately 5-fold lower than that of the parental O35E cells right after the challenge (Fig. 8C), and the O35ElpxX mutant also showed an accelerated bacterial clearance at 3 h (86.5% vs. 61.3%, p <0.01) or 6 h (96.8% vs. 88.9%, p <0.05).

Discussion

In our previous study, an lpxA gene encoding UDP-N-acetylglucosamine acyltransferase responsible for the first step of lipid A biosynthesis in M. catarrhalis serotype A strain O35E was identified and an isogenic knockout mutant exhibited with a loss of LOS structure [19]. Here, two late acyltransferase genes responsible for lipid A biosynthesis were identified and their isogenic knockout mutants were constructed. Structural analysis revealed that O35ElpxX mutant lacked two decanoic acid (C10:0) chains while O35ElpxL mutant did not acylate lipid A with a dodecanoic (lauric) acid (C12:0). In the literature, the nomenclature for the lpxL/M or htrB/MsbB is inconsistent among other bacteria. In E. coli LPS biosynthesis, the late acyltransferase lpxL was found to be responsible for the addition of a secondary laurate (C12:0) moiety to the 2′ position of lipid A [27] while an lpxM is responsible for the addition of a secondary myristate (C14:0) chain at the 3′ position of lipid A [36, 37]. In H. influenzae, the htrB (lpxL) gene product was shown to be responsible for the addition of a secondary myristate (C14:0) chain at the 2′ and 3′ positions of lipid A [31] while in meningococci, the lpxL1 (msbB) and lpxL2 were responsible for the addition of secondary laurate (C12:0) chains at the 2 and 2′ positions of lipid A [29, 38]. In our case, the lpxX stands for acylating lipid A with decanoic acids (C10:0), which has not been identified in other species. The lpxX in M. catarrhalis O35E was responsible for the addition of two secondary decanoate (C10:0) chains at the 2′ (reducing end) and 3 (non-reducing end) positions of lipid A while the lpxL was responsible for the addition of a secondary laurate (C12:0) chain at the 2 (non-reducing end) position of lipid A, suggesting that the roles of lpxX and lpxL in M. catarrhalis are not exactly the same as those of lpxL/M in E. coli, htrB (lpxL) in H. influenzae or lpxL/MsbB in meningococci at both acylation positions and secondary moieties, which match each unique LOS/LPS structure of different species [10].

With respect to the physicochemical properties, the E. coli lpxL mutation does not affect the mobility of LPS on SDS-PAGE gels, but the silver-stained LPS has a dramatically reduced intensity and a conversion from a black to a brown coloration [39]. The LOS isolated from H. influenzae strain 2019 htrB mutants migrates faster than the wild type LOS and its color changed from black to brown on the silver-stained gels [31]. Our data suggested that the migration and the staining of the LOS from O35ElpxX were similar to the patterns of H. influenzae [31] while those of the LOS from O35ElpxL were different from those of H. influenzae or E. coli [39].

The O35ElpxX that lacks two decanoic acid substituents on its lipid A was very susceptible to most hydrophobic reagents while the O35ElpxL that lacks the single dodecanoic acid substituent on its lipid A was slightly susceptible, except for deoxycholate, as compared to the parental strain. These results imply that the susceptibility of the mutants to hydrophobic reagents depends on the fatty acylation pattern of their lipid A, and that the O35ElpxX, which contains pentaacylated lipid A, allowed more diffusion of hydrophobic solutes than the O35ElpxL, which contains hexacylated lipid A. The fact that both mutants and their parental strain were resistant to a hydrophilic glycopeptide which was normally excluded by an intact enterobacterial outer membrane [40] might indicate that, even though the lipid A of the M. catarrhalis lpxL mutants are altered, their ability to have a normal OS allows them to form an outer membrane that can still resist the hydrophilic glycopeptide. It was not clear why the O35ElpxL was resistant to deoxycholate, as were the E. coli lpxL mutants [27]. The mechanism of hydrophobic reagents susceptibility in the M. catarrhalis mutants needs to be further studied.

LOS toxicity was assumed to be associated mostly with the lipid A moiety. We analyzed the toxicity of M. catarrhalis mutants by an in-vitro LAL assay. Both the C10:0 acyl chain deficient O35ElpxX and the C12:0 acyl chain deficient O35ElpxL mutants did not show reductions of the toxicity by LAL assay; however, an LOS null-mutant [19] showed decreased toxicity (0.14 EU/ml) as compared with the parental strain (3.7×10³ EU/ml). Future studies need to evaluate the toxicity of both mutants in vivo to confirm the results from the LAL assay.

In addition, the O35ElpxX was sensitive to the bactericidal activity of a normal human serum when compared to the parental strain, but less sensitive than that of the LOS-null mutant [19]. These results suggested that the permeability change on the outer membrane barrier of the M. catarrhalis mutants might increase their sensitivity to the complement killing of the serum and this permeability varied with the impairment extent of its lipid A or LOS.

In a mouse challenge model, the O35ElpxX mutant strain showed a significantly enhanced clearance than O35ElpxL mutant strain or the parental strain from mouse lungs after an aerosol challenge with viable bacteria. The difference between these two mutants in the bacterial clearance might reflect the difference in the integrity of their outer membrane, binding activity and the sensitivity of the murine complement-mediated killing.

In conclusion, the lpxX and lpxL genes responsible for two late acyltransferases, decanoyl and dodecanoyl transferases, were identified in M. catarrhalis. The acyloxyacyl linked secondary acyl chains of lipid A moiety of the LOS are important in some biological activities of M. catarrhalis. Elucidation of lipid A/LOS biosynthesis, structure and functions in vitro and in vivo may provide insights into the mechanisms of M. catarrhalis pathogenesis and the immune response to infection.

Experimental procedures

Bioinformatics

Two putative late acyltransferase genes were predicted from the partial M. catarrhalis genome (AX067448 and AX067465, NCBI patent number WO0078968). To determine the gene sequences, the putative promoter sequences were predicted by a neural network based program [41] and the ORFs of these two genes were determined with Glimmer method [42]. Topology predictions of the deduced proteins were performed using TMpred, TopPred and PredictProtein methods [43–45]. Similarities of these two proteins with late acyltransferase homologues in several other Gram-negative bacteria were searched by BLAST.

Strains, plasmids, primers and growth conditions

Bacterial strains, plasmids and primers are listed in Table 1. M. catarrhalis strains were cultured on chocolate agar plates (Remel, Lenexa, KS), or brain-heart infusion (BHI) (Difco, Detroit, MI) agar plates at 37°C in 5% CO₂. Mutant strains were selected on BHI agar supplemented with kanamycin at 20 μg/ml, zeocin 5 μg/ml or spectinomycin 15 μg/ml. Growth rates of wild type strain and mutants were measured as follows: an overnight culture was inoculated in 10 ml of BHI media (adjusted OD₆₀₀ = 0.05) and shaken at 37°C at 250 rpm. The bacterial cultures were monitored spectrophotometrically at OD₆₀₀ for 8 h. The data represented averages of three independent assays. E. coli was grown on Luria-Bertani agar plates or broth with appropriate antibiotic supplementation. The antibiotic concentrations used for E. coli were as follows: kanamycin, 30 μg/ml; zeocin, 25 μg/ml and ampicillin, 50 μg/ml.

General DNA methods

DNA restriction endonucleases, T4 DNA ligase, E. coli DNA polymerase I Klenow fragment, and Taq DNA polymerase were purchased from Fermentas (Hanover, MD). Preparation of plasmids, purification of PCR products and DNA fragments were performed using kits manufactured by Qiagen (Santa Clarita, CA). Bacterial chromosomal DNA was isolated using a genomic DNA purification kit (Promega, Madison, WI). DNA nucleotide sequences were obtained via 3070xl DNA analyzer (Applied Biosystems, Foster City, CA) and analyzed with DNASTAR software (DNASTAR Inc., Madison, WI).

Cloning of lpxX gene and construction of the knockout mutant O35ElpxX

DNA sequence containing the lpxX gene was amplified from the chromosomal DNA of M. catarrhalis strain O35E using primers b1X and b1B (Table 1, Fig. 1A). The PCR product was cloned into pCR2.1 using a TOPO TA cloning kit (Invitrogen, Carlsbad, CA) to obtain pCRlpxX. The insert was released by XhoI-BamHI digestion and then subcloned into an XhoI-BamHI site of pBluescript || SK (+) to form pSlpxX. To clone the Zeo^r gene into the lpxX gene, the lpxX PCR product was amplified from the pSlpxX using primers b1E1 and b1E2 and the Zeo^r gene was amplified from the pEM7/Zeo using primers zeo EcoRI-1 and zeo EcoRI-2, then these two PCR products were digested with EcoRI respectively, and ligated to form pSlpxX-zeo. After verification by sequence analysis, the disrupted lpxX gene containing the inserted Zeo^r gene in pSlpxX-zeo was amplified by PCR and purified for electroporation to O35E competent cells as described previously [19]. After 24 h incubation, the resulting Zeo^r colonies were selected for PCR identification using primers b1X and b1B and the inactivated lpxX mutant was verified by sequencing.

Cloning of lpxL gene and construction of the knockout mutant O35ElpxL

DNA sequence containing the lpxL gene was amplified from chromosomal DNA of strain O35E using primers b2E and b2B (Table 1, Fig. 1B), and cloned into pCR2.1 using a TOPO TA cloning kit to obtain pCRlpxL. The insertion was released by EcoRI-BamHI digestion, and then subcloned into an EcoRI-BamHI site of pBluescript || SK (+) to form pSlpxL. The Kan^r cassette (1240 bp) obtained from pUC4K after PstI digestion was subsequently cloned into the lpxL gene using a PstI site to form pSlpxL-kan. After verification by sequence analysis, the disrupted lpxL gene with the inserted Kan^r gene in pSlpxL-kan was amplified by PCR using primers b2E and b2B. The PCR product was purified and used for electroporation to O35E competent cells [19]. The resulting Kan^r colonies were selected for PCR analysis using primers b2E and b2B and the inactivated lpxL mutant was verified by sequencing.

Southern blot

The Zeo^r gene or Kan^r gene was amplified from pEM7/Zeo or pUC4K as a probe with primers zeo EcoRI-1/zeo EcoRI-2 or kan RP/kan FP (Table 1) by using a PCR digoxigenin (DIG) probe synthesis kit (Roche, Indianapolis, IN). Southern blot analyses of the chromosomal DNA from strains O35E, O35ElpxX and O35ElpxL were performed using a DIG DNA labeling and detection kit (Roche) according to the instruction manual. The hybridization temperature of the Southern blot was at 42°C and the blots were washed under high stringency condition (65°C in 0.5 × SSC with 0.1% SDS).

RT-PCR

Total RNA was isolated from log-phase bacteria of each strain using an RNeasy Mini kit and treated with an on-column RNase-Free DNase set (Qiagen). The first-strand synthesis of cDNA was primed with random primers using a high capacity cDNA archive kit (Applied Biosystems). Primer sets for PCR amplification of target genes ats, lpxX and lgt 6 in cDNA samples were ats1/ats2, b1SP/b1AP, and lg1/lg2, respectively (Table 1, Fig. 1A). Primer sets for PCR amplification of target genes atr, lpxL, and asd in cDNA samples were atr1/atr2, b2SP/b2AP, and asd1/asd2, respectively (Table 1, Fig. 1B). In parallel, PCRs were performed with chromosomal DNA as positive controls and cDNA samples without activation of the reverse-transcription reaction as negative controls. The PCR products were resolved on 0.8% agarose gels and visualized by ethidium bromide staining.

Reversion of O35ElpxX and O35ElpxL mutants

Primers b1B1/b1S or b2B1/b2S (Table 1; Fig. 1) were used to amplify native lpxX or lpxL gene from chromosomal DNA of the wild type strain O35E. The resulting PCR products were purified and subcloned into pWW115 plasmid [47] using BamHI and SacI, and the constructs were transformed into O35E competent cells by electroporation [47]. The bacteria were plated onto BHI agar containing 15μg/ml spectinomycin. Plasmids were extracted from the spectinomycin-resistant colonies, identified by digestion with BamHI and SacI as well as by sequence analysis, and named as pWlpxX and pWlpxL. The pWlpxX and pWlpxL were used to transform O35ElpxX and O35ElpxL competent cells by electroporation, and the resulting cell suspensions were plated onto BHI agars containing spectinomycin. Potential revertant colonies were identified and chosen for further analyses.

LOS determination

A crude LOS extraction was performed from strain O35E, O35ElpxX or O35ElpxL using a method of proteinase-K treated whole cell lysates [48]. The resulting extracts from each bacterial suspension (1.9 μg of protein concentration) were resolved by 15% SDS-PAGE and visualized by silver staining [49].

Structural analysis of lipid A

The LOS from 30–35g of wet cells of each strain was prepared by phenol-water extraction [50]. Lipid A was released from each LOS using the SDS mild acid hydrolysis method [51]. Briefly, LOS samples were hydrolyzed in 10 mM NaOAc in 1% SDS, pH 4.5 buffer at 100°C for 1.5 h, with constant stirring. Hydrolysates were then freeze-dried, washed with 95% acidified ethanol, and centrifuged at 3500 rpm. Obtained sediments were washed twice with ethanol, following by centrifugation, then re-suspended in water and lyophilized. The remaining traces of oligosaccharides and SDS were removed by threefold extraction with chloroform:water (1:1). Organic phases, after concentration, were used for composition and MS analyses.

Lipid A structure was analyzed by MALDI-TOF MS. Spectra of the lipid A preparations were acquired using Applied Biosystems 4700 Proteomics System Spectrometer in reflector mode. Samples were dissolved in a chloroform:methanol mixture (3:1) and 1μl was then mixed with 1μl of the 0.5 M 2,4,6-trihydroxyacetophenone monohydrate (THAP) matrix in methanol and spotted onto the stainless steel MALDI plate. Spectra were recorded in negative ion mode.

The fatty acid compositions of the lipid A preparations were done by the preparation of FAMEs and GC-MS analysis. The FAMEs were obtained by methanolysis of lipid A using methanolic 1M HCl at 80°C for 18 h. The FAMEs were recovered into organic phase by a threefold extraction with 50% NaCl:chloroform (1:1 v/v). A new volume of chloroform was used for each of the three extractions. Finally the organic phase was re-extracted with deionized water to remove traces of salts, and the volume of the organic phase was reduced prior GC-MS analysis. Analysis was performed on a Hewlett-Packard HP5890 gas chromatograph equipped with a mass selective detector 5970 MSD. A DB-1 fused silica capillary column (30 m×0.25 mm I.D.; J & W Scientific, Folsom, CA) was used for this analysis with helium as the carrier gas. The temperature program was: 70°C for 2 min, then ramping to 220°C at 8°C/min with a 10 min hold, then ramping to 280°C at 10°C/min.

Structural analysis of oligosaccharides (OSs)

For OS analysis, core OSs were released from LOS by mild hydrolysis with 1% (v/v) HOAc at 100°C for 1.5 h and further purified using Bio-Gel P-2 gel-filtration chromatography with water as the eluant. The eluting fractions were monitored with Shimadzu Refractive Index Detector (RID-10A). Purified OSs were analyzed by MALDI-TOF MS using AB Proteomics Analyzer 4700 (Applied Biosystems). The OS samples were dissolved in nanopure water and mixed with 0.5 M 2,5-dihydroxy benzoic acid matrix in methanol in 1:1 (v/v) ratio. Samples were then applied onto a stainless steel MALDI plate and spectra were acquired in a positive reflector mode.

Limulus amebocyte lysate (LAL) assay

The chromogenic LAL assay for endotoxin activity was performed using the QCL-1000 kit (Bio-Whittaker Inc., Walkersville, MD). Overnight cultures of the parental strains and two derived mutants from chocolate agar plates were suspended in BHI broth to OD₆₂₀ of 0.1 and serial dilutions of these stocks were tested based on the instruction of the manufacture.

Susceptibility determination

The sensitivity of strains to a panel of hydrophobic agents or a hydrophilic glycopeptide was performed using standard disk-diffusion assays [52]. Bacteria were cultured in BHI to an OD₆₀₀ of 0.2 and 100 μl portions of the bacterial suspension were spread onto chocolate agar plates. Antibiotic disks or sterile blank paper disks (6 mm, Becton Dickinson, Cockeysville, MD) containing the various agents were plated on the lawn in triplicate at 37°C for 18 h. Sensitivity was assessed by measuring the diameter of the zone of growth inhibition in two axes and the mean value was calculated.

Bactericidal assay to normal human serum

A complement-sufficient normal human serum was prepared and pooled from 8 healthy adult donors. A bactericidal assay was performed in a 96-well plate [18]. Normal human serum was diluted to 0.5, 2.5, 12.5, and 25% in pH 7.4 Dulbecco’s phosphate-buffered saline containing 0.05% gelatin (DPBSG). Bacteria [10 μl containing 10⁶ CFU (colony-forming units)] were inoculated into 190 μl reaction wells containing the diluted normal human serum, 25% of heat-inactivated normal human serum, or DPBSG alone and incubated at 37°C for 30 min. Serial dilutions (1:10) of each well were plated onto chocolate agar plates. The resulting colonies were counted after 24 h of incubation.

Pulmonary clearance patterns in animal model

Female BALB/c mice (6–8 weeks of age) were obtained from Taconic Farms, Inc. (Germantown, NY). The mice were housed in an animal facility in accordance with National Institutes of Health guidelines under animal study protocol 1158–04. Bacterial aerosol challenges were carried out in mice using the OD₅₄₀ of 0.4 for wild type strain O35E (1.3×10⁹ CFU/ml) or the OD₅₄₀ of 0.43 for the mutant O35ElpxX (1.0×10⁹ CFU/ml) or the A₅₄₀ of 0.4 for the O35ElpxL (1.8×10⁹ CFU/ml) in a 10 ml DPBSG [53]. The number of bacteria present in the lungs was measured at various time points post-challenge. The minimum detectible number of viable bacteria was 100 CFU per lung. Clearance of M. catarrhalis was expressed as the percentage of bacterial CFU at each time point compared with the number deposited at time zero.

Statistical analysis

The number of viable bacteria were expressed as the geometric mean CFU of eight (mice) independent observation ± standard deviation (SD). The significance of the clearance rate was analyzed by a Chi-square test (two tailed). One-way ANOVA was employed for multiple point comparison.

Nucleotide sequence accession number

The nucleotide sequences of the lpxX and lpxL genes in M. catarrhalis strain O35E were deposited at GenBank under accession number EU155137 and EU155138, respectively.

Abbreviations

LOS

lipooligosaccharide

LPS

lipopolysaccharide

OS

oligosaccharide

Kdo

3-deoxy-D -manno-octulosonic acid

BHI

brain-heart infusion

Zeo^r

zeocin-resistant

Kan^r

kanamycin-resistant

FAMEs

fatty acid methyl esters

LAL

Limulus amebocyte lysate

EU

endotoxin units

CFU

colony-forming units