Identification of Mycobacterium avium genes associated with resistance to host antimicrobial peptides

Abstract

Antimicrobial peptides are an important component of the innate immune defence. Mycobacterium avium subsp. hominissuis (M. avium) is an organism that establishes contact with the respiratory and gastrointestinal mucosa as a necessary step for infection. M. avium is resistant to high concentrations of polymyxin B, a surrogate for antimicrobial peptides. To determine gene-encoding proteins that are associated with this resistance, we screened a transposon library of M. avium strain 104 for susceptibility to polymyxin B. Ten susceptible mutants were identified and the inactivated genes sequenced. The great majority of the genes were related to cell wall synthesis and permeability. The mutants were then examined for their ability to enter macrophages and to survive macrophage killing. Three clones among the mutants had impaired uptake by macrophages compared with the WT strain, and all ten clones were attenuated in macrophages. The mutants were also shown to be susceptible to cathelicidin (LL-37), in contrast to the WT bacterium. All but one of the mutants were significantly attenuated in mice. In conclusion, this study indicated that the M. avium envelope is the primary defence against host antimicrobial peptides.

Introduction

Mycobacterium avium subsp. hominissuis (hereafter M. avium) is a pathogen that infects humans and other mammals by crossing mucosal barriers. In humans, M. avium causes disease in both immunocompromised and immune-competent individuals (Ashitani et al., 2001; Marras & Daley, 2002). Indication of infection in most of the patients is connected to signs and symptoms associated with the respiratory tract or systemic disease (Ashitani et al., 2001; Marras & Daley, 2002).

In order to cause infection by crossing the respiratory and intestinal mucosas, M. avium has to resist the action of antimicrobial peptides present on mucosal surfaces. Human intestinal mucosa secretes β-defensins, cathelicidin and Reg IIIβ (Bevins & Salzman, 2011; Ouellette, 1999), while chiefly cathelicidin and defensins are found in the respiratory tract mucosa (Bals, 2000). Antimicrobial peptides play an important role in the host innate response against a number of bacteria (Sørensen et al., 2008) and perhaps against mycobacteria.

High concentrations of antimicrobial peptides are encountered in the mucus layer, preventing bacteria from moving closer to the mucosal surface. In the intestinal lumen, M. avium has to cross two layers of mucus, one of them with large concentrations of antimicrobial peptides (Johansson et al., 2011). In fact, work by Johansson’s group has shown that while the external mucus layer contains many bacteria and a low concentration of antimicrobial peptides, the innermost layer of mucus is rich in antimicrobial peptides and generally deficient in micro-organisms (Johansson et al., 2008). M. avium does not have flagella or any other mechanisms to move across the mucus layers towards the mucosa. Therefore, in order for the bacterium to establish contact with the mucosal surface, it should possess a mechanism that would confer resistance to the harmful environment of the mucus, perhaps for an extended period of time. We now have evidence that M. avium does not bind to mucin, which may facilitate the bacterial migration in the mucus (J. Bechler, K. Gilbert & L. E. Bermudez, unpublished results). However, the pathogen should also be able to resist the action of antimicrobial peptides.

Prior studies have suggested that Mycobacterium tuberculosis may be susceptible to rabbit and human defensins and to other antimicrobial peptides (Miyakawa et al., 1996). In addition, other studies have also shown that neutrophil proteins, HNP-1, 2 and 3, have bactericidal activity against organisms of the M. avium complex (Ogata et al., 1992). More recently, it has been shown that Mycobacterium avium subsp. paratuberculosis, a species closely related to M. avium subsp. hominissuis, resists the action of several antimicrobial peptides (Alonso-Hearn et al., 2010).

Maloney and colleagues observed that M. tuberculosis cell surface phospholipids are in their majority lysinylated (Maloney et al., 2009). The authors also demonstrated that in the absence of the lysX gene (involved in the lysinylation of surface phospholipids), the bacterium becomes susceptible to antimicrobial peptides in vitro and is attenuated, compared with the WT bacterium in vivo (Maloney et al., 2009). The findings indicate that the susceptibility of pathogenic mycobacteria to antimicrobial peptides depends upon the components on the bacterial surface, the expression of which can vary depending on the environment. PhoP, a major regulator of the cell wall in many bacterial species, including M. tuberculosis, is associated with the resistance to antimicrobial peptides as well (Ryndak et al., 2008).

Antimicrobial peptides have been shown to possess antibacterial, antifungal and antiviral activities in model systems in vitro (Carroll et al., 2010). Antimicrobial peptides are also produced by activated phagocytes and previous work has demonstrated their role in the phagocyte’s mechanisms of killing (Brogden, 2005).

Macrophages and neutrophils contain antimicrobial peptides that participate in the killing of intracellular bacteria. The bactericidal molecules are delivered from the lysosome into the pathogen’s vacuole, where they can be inserted in the bacterial cell envelope (Duplantier & van Hoek, 2013). Because virulent mycobacteria inhibit the fusion of the phagosome with the lysosome, most of the bactericidal peptides are probably not delivered to the bacterial environment (Sturgill-Koszycki et al., 1994).

M. avium comes in contact with both the mucosal surface and phagocytic cells. Therefore we initiated the investigation on the resistance of the pathogen to antimicrobial peptides by screening a transposon library. Our screen was able to identify several genes associated with resistance to antimicrobial peptides, and allowed us to begin to understand how the bacterium can defend itself against the action of powerful bactericidal molecules.

Methods

Bacterial strains and growth conditions.

Mycobacterium avium subsp. hominissuis 104 is a virulent strain isolated from the blood of an AIDS patient. The bacterium was cultured on Middlebrook 7H11 agar supplemented with 10 % oleic acid, albumin, glucose and catalase (OADC; Difco Laboratories) or grown in Middlebrook 7H9 broth enriched with 0.2 % glycerol and OADC (Difco Laboratories). Bacteria were used in the exponential phase as well as in the stationary phase of growth.

Escherichia coli DH5α (Stratagene) was grown on Luria–Bertani broth or an agar plate (Difco Laboratories). Antimicrobials were added to the culture media to give the following concentrations: 32 µg polymyxin B ml⁻¹ and 200 µg kanamycin ml⁻¹).

MIC determination.

The inoculum was prepared by picking up five to ten colonies (only transparent colonies) from a 7H10 agar plate. The colonies were transferred into 7H9 broth and allowed to grow for 24 h. The MICs were determined by seeding 10⁵ c.f.u. of Mycobacterium avium complex (MAC) 104 mutants or the WT bacterium into 96-well round-bottom plates in the presence of Middlebrook 7H9 broth, 10 % OADC and serial dilutions of polymyxin B. Controls included undiluted inoculum without drug, and inoculum diluted 1 : 10 without drug. After 5 and 10 days of incubation at 37 °C, turbidities were compared with the controls with no antibiotic. Samples were plated onto 7H10 agar plates to confirm the results. Significant activity was defined as a reduction of two or more orders of magnitude over the period of the test.

M. avium transposon library and screening.

An M. avium 104 transposon library was constructed as previously described (Li et al., 2010). It was duplicated into 96-well flat-bottomed tissue culture plates to test for susceptibility to polymyxin B (Sigma), a cationic antimicrobial peptide surrogate. The MIC of polymyxin B for M. avium was found to be >500 µg ml⁻¹. Bacterial clones were then exposed to 32 µg ml⁻¹ of polymyxin B and incubated at 37 °C. Growth was measured after 7 days of incubation by comparing the turbidity of wells with or without polymyxin B. Clones showing susceptibility (no growth) in the presence of polymyxin B were retested by plating onto 7H10 agar plates to confirm the phenotype.

Sequencing of clones and analysis.

Genes interrupted in identified clones were sequenced using the method previously described by Danelishvili et al. (2007). DNA sequences were obtained at the Centre for Gene Research and Biocomputing, Oregon State University. Database search and analysis were performed using blast (Basic Local Alignment Search Tool). The M. avium sequence DNA from the National Centre for Biotechnology Information (NCBI) database was used to confirm the sequences obtained.

Susceptibility to cathelicidin (LL-37).

Purified LL-37 peptide (Ana Spec) was used at concentrations of 5 and 10 µg ml⁻¹ in PBS. Bacteria were exposed to both concentrations of LL-37 at 37 °C for 3 h and an aliquot of the suspension was removed and plated onto 7H10 plates for quantification of the number of colonies.

Macrophage assay.

To determine the susceptibility to macrophage killing, WT bacterium and clones were incubated with THP-1 macrophage monolayers containing 1×10⁵ cells (previously stimulated with 50 µg phorbol ester ml⁻¹) at an m.o.i. of five. Phagocytosis was allowed to happen at 37 °C for 2 h in the presence of RPMI-1643 medium containing heat-inactivated foetal calf serum. Then the monolayers were washed with Hank’s buffered salt solution. The contents of some wells were then lysed with sterile water to release viable intracellular bacteria. The lysate suspension was serially diluted and then plated onto 7H11 agar plates to quantify the number of viable bacteria. After establishing the number of bacteria taken by macrophage monolayers at 2 h, the remaining monolayers were followed for 4 days either without stimulation or with stimulation with 100 U of 1,25-dihydroxyvitamin D3 (1,25 vit D3). Monolayers were then lysed using the same procedure described above. The number of c.f.u. at day 4 was compared with the number of c.f.u. at 2 h after infection to determine the increase or reduction in the number of bacteria.

In vivo virulence assessment.

C57/BL6 bg+/bg− mice were infected via the caudal vein with approximately 3×10⁷ bacteria (MAC 104 and mutants). Some mice were sacrificed 1 week post-infection to establish the baseline level of bacteria in the spleen and liver. The remaining mice were sacrificed 4 weeks after infection. Splenic and hepatic tissues were removed and homogenized in 3 and 5 ml, respectively, of 7H9 broth containing 20 % glycerol. Spleen and liver homogenates were serially diluted and plated onto 7H11 agar plates which were then incubated at 37 °C. After 10 days, bacterial infection load was determined by counting the c.f.u. A total of ten mice per experimental group and seven mice for the 1 day inoculum determination were used.

Statistical analysis.

A Student’s t-test was employed to compare experimental groups and controls in all the experiments. A P value <0.05 was considered statistically significant.

Results

Susceptibility in vitro

A transposon library was screened in vitro against polymyxin B, a surrogate for antimicrobial peptides, for clones susceptible to 32 µg ml⁻¹. Approximately 2400 clones were evaluated for susceptibility to polymyxin B. The WT M. avium showed significant resistance to antimicrobial peptides and polymyxin B, with an MIC greater than 500 µg ml⁻¹. Ten mutants exhibited a three or more fold reduction in growth in the presence of 32 µg polymyxin B ml⁻¹. All ten mutants failed to show increased susceptibility to sub-inhibitory concentrations of isoniazid (INH) and clarithromycin but were more susceptible to ethambutol (data not shown).

Identification of mutations in M. avium clones

Transposon insertion locations were elucidated by amplifying flanking regions of Tn5367, as previously reported (Danelishvili et al., 2007; Li et al., 2010). Table 1 lists all the mutants identified. All the genes interrupted either play a role in generic cellular function, or encode hypothetical proteins that are upstream to genes involved in cell wall permeability, or are directly involved in cell wall permeability or fatty acid biosynthesis.

Table 1. Genes identified in the M. avium mutants associated with susceptibility to polymyxin B.

Mutant no.	Gene	Accession no.	Function
1	MAV_4265	ABK69290	Aldehyde dehydrogenase (NAD) family protein
2	MAV_3253	YP_882435	Hypothetical protein from the daunorubicin resistance gene cluster
3	MAV_0119	YP_879415	Thiopurine S-methyltransferase (tpmt)
4	MAV_0216	ABK65610	Cutinase superfamily protein
5	MAV_3616	ABK66306	Long-chain specific acyl-CoA dehydrogenase
6	MAV_4687	ABK68276	Dihydrolipoamide dehydrogenase
7	MAV_3373	YP_882794	Methyltransferase, UbiE/COQ5 family protein
8	MAV_3210	YP-882392	Glycogen debranching enzyme GlgX
9	MAV_2191	ABK67230	β-Ketoacyl-acyl carrier protein (ACP) synthase (KAS), type II
10	MAV_2450	YP_881643	Erythronolide synthase (polyketide synthase), modules 3 and 4

Inactivation of KasB (MAV_2191, mutant number nine) results in the synthesis of mycotic acids that are two to four carbons shorter than the mycolic acid in the WT bacterium (Gao et al., 2003). KasB inhibition strikingly increases cell wall permeability to lipophilic compounds but has shown little effect on resistance to hydrophilic compounds (Gao et al., 2003).

MAV_0119, a gene interrupted in mutant number three, encodes a hypothetical protein that shows similarities to phosphatidylethanolamine N-methyltransferase, a principal component of cell membranes.

Mutants one, five, six and seven are deficient in genes involved in cell wall synthesis. Tn5367 was located in MAV_0216, a gene encoding a hypothetical protein. This protein has similarity with the cutinase superfamily and analysis of the surrounding genes suggests that the transposon may have interrupted an operon, thereby suppressing the upstream genes which are associated with cell wall permeability (acyl-GA synthase, polyketide synthase, acetyl/propionyl-coenzyme A carboxylase β unit).

The transposon also interrupted a polyketide synthase (PKS), analogue to the Mycobacterium avium subsp. paratuberculosis PKS 12 and M. tuberculosis PKS 12. The PKS 12 is involved in the synthesis of phthiocerol dimycocerosate, a major cell wall lipid. Phthiocerol dimycocerosate is an integral element of the cell wall of pathogenic mycobacteria and has been hypothesized to make the cell wall impermeable (Camacho et al., 2001).

Macrophage uptake and killing

To determine whether alterations in the bacterial cell envelope had any impact on uptake and survival in macrophages, we examined the interaction of the clones with macrophages. As shown in Table 2, three of the clones tested had a significant decrease in the ability to infect macrophages, although not all the mutations were associated with an impact on phagocytosis. Three of the mutants had their uptake by macrophages impaired at 30 min while for two of the clones, the phenotype was still observed at a later time point when compared with the uptake of the WT strain.

Table 2. Macrophage infection assay with infection at 30 min and 2 h comparing the WT M. avium 104 and mutant clones.

		Phagocytosis
	Strain/gene	30 min	2 h
	MAC 104 (WT)	4.9±0.4×105	8.2±0.3×105
1	(4C8)/MAV_4265	3.4±0.4×105	7.1±0.5×105
2	(6E10)/MAV_3253	5.3±0.5×105	8.8±0.6×105
3	(7D10)/MAV_0119	5.4±0.6×105	8.4±0.6×105
4	(2G6)/MAV_0216	8.8±0.3×104*	6.9±0.4×105
5	(36H10)/MAV_3616	5.6±0.4×104*	2.3±0.4×104*
6	(23B4)/MAV_4687	5.0±0.6×105	8.4±0.4×105
7	(2H2)/MAV_3373	5.8±0.6×105	8.0±0.5×105
8	(25E10)/MAV_3210	4.4±0.5×105	7.5±0.7×105
9	(2C6)/MAV_2191	4.9±0.6×105	8.2±0.6×105
10	(1C2)/MAV_2450	5.3±0.6×104*	3.1±0.4×105*

^*P<0.05 compared with M. avium 104 WT.

In Table 3, the results demonstrate that all of the clones were attenuated in non-stimulated macrophages. While some of the clones were still able to replicate within macrophages, four of the mutants had a significant decrease in the number of intracellular bacteria compared with the WT bacterium and with the number of intracellular bacteria at the time after infection.

Table 3. Macrophage survival assay comparing the ability of the WT M. avium 104 with mutant clones.

		No. c.f.u. per 105 macrophage lysate
	Strain/gene	1 h	4 days (with 1,25 vit D3)	4 days (without 1,25 vit D3)	Outcome
	MAC 104 (WT)	6.1±0.3×105	5.7±0.4×104	3.0±0.4×106
1	(4C8)/MAV_4265	3.4±0.4×105	1.6±0.3×104	6.2±0.3×105*	Impaired
2	(6E10)/MAV_3253	3.3±0.5×105	8.9±0.5×103	5.4±0.4×105*	Impaired
3	(7D10)/MAV_0119	5.9±0.5×105	2.1±0.5×105	7.9±0.4×105*	Impaired
4	(2G6)/MAV_0216	4.6±0.7×104	7.3±0.4×104	2.3±0.5×105*	Decreased
5	(36H10)/MAV_3616	2.3±0.4×105	3.3±0.6×104	4.0±0.3×105*	Impaired
6	(23B4)/MAV_4687	3.7±0.3×105	1.1±0.4×105	7.3±0.5×105*	Impaired
7	(2H2)/MAV_3373	3.8±0.5×105	5.0±0.3×104	2.9±0.3×105*	Decreased
8	(25E10)/MAV_3210	2.1±0.5×105	6.9±0.5×104	5.1±0.7×105*	Impaired
9	(2C6)/MAV_2191	1.6±0.4×105	2.2±0.4×104	8.4±0.3×104*	Decreased
10	(1C2)/MAV_2450	2.0±0.6×104	1.7±0.3×103	6.0±0.3×103*	Decreased

^*P<0.05 comparing the growth of the mutant strain with the WT growth.

It is also of note that when macrophages were stimulated with 1,25 vit D3, the killing of intracellular M. avium strains increased substantially. 1,25 vit D3 induces the synthesis of cathelicidin by macrophages (Liu et al., 2006). Past work has demonstrated that the killing of M. avium in macrophages following stimulation with 1,25 vit D3 is due to the secretion of TNF-α, granulocyte-macrophage colony-stimulating factor and antimicrobial peptides (Bermudez et al., 1990).

Susceptibility to LL-37 (cathelicidin)

Humans, in contrast to many other mammals, have only one cathelicidin gene, and its expression leads to bactericidal activity in many tested systems. To evaluate if cathelicidin had comparable activity to polymyxin B, we exposed the WT bacterium and the transposon mutants obtained to different concentrations of LL-37 and determined the number of viable bacteria after 3 h. Almost all the mutants showed susceptibility to 5 µg ml⁻¹, while the WT bacterium apparently resisted the bactericidal effect of LL-37. All of the mutants were susceptible to 10 µg cathelicidin ml⁻¹ (Table 4).

Table 4. Activity of LL-37 against M. avium 104 and mutant clones.

Bacteria were exposed to 5 µg ml⁻¹ or 10 µg ml⁻¹ recombinant LL-37 for 3 h and then plated onto 7H10 agar.

		LL-37 concentration
	Strain/gene	Inoculum	5 µg ml−1	10 µg ml−1
	MAC 104 (WT)	2.4±0.3×104	2.6±0.3×104	2.3±0.4×104
1	(4C8)/MAV_4265	3.1±0.4×104	2.1±0.4×104	1.0±0.3×104*
2	(6E10)/MAV_3253	2.6±0.2×104	2.6±0.4×104	9.3±0.2×103*
3	(7D10)/MAV_0119	2.5±0.3×104	2.0±0.4×104	8.9±0.5×103*
4	(2G6)/MAV_0216	2.8±0.3×104	9.7±0.5×103*	6.1±0.3×103*
5	(36H10)/MAV_3616	2.7±0.2×104	1.2±0.2×104*	7.8±0.2×103*
6	(23B4)/MAV_4687	3.0±0.4×104	1.4±0.3×104*	6.7±0.4×103*
7	(2H2)/MAV_3373	3.1±0.4×104	1.1±0.3×104*	7.5±0.4×103*
8	(25E10)/MAV_3210	2.6±0.2×104	8.1±0.5×105*	5.3±0.6×103*
9	(2C6)/MAV_2191	2.9±0.3×104	9.6±0.5×104*	6.4±0.3×103*
10	(1C2)/MAV_2450	2.7±0.4×104	9.8±0.3×104*	5.9±0.5×103*

^*P<0.05 compared with the WT M. avium 104 control.

In vivo studies

To examine whether the mutations in M. avium led to attenuation in vivo, C57/BL-6 mice were infected with the bactericidal strains intravenously. At week 4 after infection, the spleen and liver of the mice were harvested and the number of bacteria per organ was determined. As displayed in Table 5, only mutant number five (inactivation of MAV_3616) did not show attenuation in vivo. All other tested mutants were attenuated. Mutant numbers two, three, nine and ten had severe impairment of virulence as demonstrated by a significant decrease in colony counts in both the spleen and liver of mice.

Table 5. Evaluation of virulence of the mutations in comparison to the WT M. avium 104 in C57 BL/6 mice.

		No. c.f.u. per organ*
	Mutant gene	1 day		4 weeks
		Liver	Spleen	Liver	Spleen
	MAC 104 (WT)	2.6±0.4×105	3.8±0.5×105	4.2±0.6×106	7.4±0.5×107
1	(4C8)/MAV_4265	−	−	3.2±0.3×104†	1.0±0.6×104†‡
2	(6E10)/MAV_3253	−	−	<2.0×102‡	<2.0×102‡
3	(7D10)/MAV_0119	−	−	1.4±0.3×104†	5.6±0.5×103†‡
4	(2G6)/MAV_0216	−	−	2.9±0.4×105†	3.8±0.5×105†
5	(36H10)/MAV_3616	−	−	3.1±0.5×106	8.2±0.3×107
6	(23B4)/MAV_4687	−	−	1.9±0.6×106†	6.0±0.4×106†
7	(2H2)/MAV_3373	−	−	8.8±0.4×105†	4.1±0.6×106†
8	(25E10)/MAV_3210	−	−	1.7±0.5×105†	1.2±0.5×105†
9	(2C6)/MAV_2191	−	−	3.7±0.3×104†‡	2.1±0.6×102†‡
10	(1C2)/MAV_2450	−	−	4.1±0.3×104†‡	2.7±0.3×102†‡

−, Not determined.

^*The results represent the mean±SEM.

^†P<0.05 compared with M. avium WT control at 4 weeks post-infection.

^‡P<0.05 compared with WT control at 1 day (inoculum determination).

Discussion

The innate immunity plays an important role in detecting and eradicating pathogens, although the details of the complex interactions between players remain incompletely known (Brogden et al., 2003). Studies have stressed the importance of epithelial-derived as well as phagocyte-expressed antimicrobial peptides; observations in mice deficient in genes encoding cathelicidin confirmed the increased susceptibility to infections (Brogden et al., 2003; van der Does et al., 2012).

Mycobacteria are a group of pathogens that infect many host cells but preferentially macrophages. Mycobacteria, therefore, must have a significant number of strategies to be able to cause disease in mammals. One of the mechanisms used by the host to eliminate pathogens is the production of antimicrobial peptide molecules that are released both on the mucosal surfaces and intracellularly in phagocytic cells (Becknell et al., 2013; Hansdottir et al., 2008; van der Does et al., 2012). Studies in the past have demonstrated that human defensins have bactericidal and/or bacteriostatic activity in vitro against M. avium (Ogata et al., 1992; Shin & Jo, 2011) and M. tuberculosis (Miyakawa et al., 1996; Rivas-Santiago et al., 2006; Shin & Jo, 2011). In addition, more recent observations have supported the activity of cathelicidin (LL-37) against M. tuberculosis (Rivas-Santiago et al., 2006, 2008; Sonawane et al., 2011; van der Does et al., 2012). Cathelicidin expression in humans can be stimulated by the presence of 1,25 vit D3, and a number of studies have shown evidence that M. tuberculosis and M. avium infections can be attenuated by controlling bacterial replication in macrophages following stimulation by 1,25 vit D3 (Bermudez et al., 1990; Rivas-Santiago et al., 2008; Yuk et al., 2009). In contrast, other groups have been less successful in establishing the correlation between M. tuberculosis survival and antimicrobial peptide production (Rivas-Santiago et al., 2006; Sow et al., 2011). In fact, work by Maloney et al. (2009) described a mutation in the LysX protein of M. tuberculosis, a lysyl-transferase synthetase, which makes the bacterium susceptible to the action of antimicrobial peptides, suggesting that in conditions in which the protein is expressed and lysinylation occurs, the bacterium is potentially resistant to antimicrobial peptide molecules.

M. avium is even more resistant to antibiotics than M. tuberculosis and because it has the ability to survive in harsh environments as well as within environmental hosts containing a diverse array of killing mechanisms (Inderlied et al., 1993), it is assumed to have a cell wall which is harder to penetrate. To improve the understanding about susceptibility to antimicrobial peptides, we decided to screen a transposon bank of mutants against the action of polymyxin B, a surrogate for bactericidal peptides, and to test the identified mutants with increased susceptibility to the antimicrobial in a number of model systems in vitro and in vivo. The results of this study indicated that inactivation of cell wall synthesis/maintenance-related genes leads to susceptibility to antimicrobial peptides, and in the majority of the mutant strains, a reduction in the ability to cause attenuation in macrophages and in mice. Interestingly, three of the mutations were associated with a reduction in uptake by macrophages at 30 min and 2 h. This observation has two implications. Firstly, because the phagocytosis assay was carried out in the absence of opsonizing components of the serum, the results indicate that alterations in the bacterial cell wall may impair uptake and make the bacteria more difficult to be ingested by phagocytes. The fact that fewer viable bacteria were isolated from macrophages at 1 h may indicate that they were killed upon uptake. The other implication is that mutant bacteria may enter macrophages by a pathway which is not the usual ‘pathogen-related’ pathway, therefore increasing the likelihood that they will be subject to the phagocyte bactericidal arsenal. However, we could not demonstrate by inhibiting rapid mechanisms of killing (superoxide- and nitric oxide-dependent) that this had any effect on the number of intracellular bacteria (data not shown).

Antimicrobial peptides are small molecules produced and secreted by epithelial cells and phagocytes (van der Does et al., 2012). Many studies have demonstrated that mycobacterium infection results in increased production of the bactericidal molecules, including cathelicidin (Rivas-Santiago et al., 2008; Shin & Jo, 2011). More recently, it has been shown that LL-37 regulates the transcription of autophagy-related genes, such as beclin-1 and atg 5, and still other macrophage functions, suggesting that it does not only have direct antibacterial activity but also participates actively in the activation and regulation of other innate immune functions. The macrophages, 1,25 Vit D3 and cathelicidin are involved in the killing of pathogens (Yuk et al., 2009). In addition, M. tuberculosis (but not M. avium) killing in macrophages has been linked to autophagy (Gutierrez et al., 2004).

When exposed to sub-inhibitory concentrations of clarithromycin and INH, none of the mutants identified in our work showed an increased susceptibility to the antibiotics. However, they were all more susceptible to ethambutol (data not shown). This observation may correlate with the particular action of ethambutol on the cell wall of M. avium (Mikusová et al., 1995) or it may be that the bacterial cell wall works as a partial barrier to the compound.

Based on the results of our study, interference with mycolic acid synthesis (synthesis of dimycocerosyl phthiocerol, a major cell wall lipid, which has been associated with cell wall permeability and other genes linked to cell wall synthesis) enhanced susceptibility to antimicrobial peptides. M. avium probably faces the challenge of antimicrobial peptides in the mucosal surface. It is plausible to speculate that M. avium, when in the intestinal tract environment, contains a cell envelope that is resistant to antimicrobial peptides. In macrophages, however, because M. avium is able to inhibit phagosome–lysosome fusion (Sturgill-Koszycki et al., 1994), the contact with antimicrobial peptides may not occur in principle, or at least not for all the intracellular bacteria. To explain the increased susceptibility observed both in macrophages and in vivo, one must consider the fact that other pathways, such as autophagy, may contribute to the attenuation observed. Alternatively, some of the attenuation observed with mutants when in macrophages and in mice may be explained by a combination of factors in addition to the inability to inhibit phagosome–lysosome fusion. Therefore, the mechanism of susceptibility of the mutants in macrophages is probably multifold. A mutant deficient in polyketide synthase has been previously described, but an association with superoxide anion or nitric oxide production by macrophages has not been established (Li et al., 2010). In fact, the mechanisms associated with the macrophage killing of organisms belonging to the M. avium complex are poorly understood. Mutant number eight, with inactivation of an oligosyltrehalose synthase, may illuminate a possible mechanism since several micro-organisms respond to environmental stresses by accumulating high levels of trehalose (Zaragoza et al., 2003). Trehalose is the only detectable free sugar in mycobacteria. Inability to respond properly to environmental stresses and challenges may explain, in part, the susceptibility of this particular mutant in both macrophages and mice.

The mutant number five, although attenuated in the macrophage assay, did not show attenuation in vivo. Although it was unexpected, it may occur, for example, if any of the host environments stimulate the synthesis of an enzyme that can substitute for the function of the Acyl-CoA dehydrogenase.

In summary, by screening a transposon library for increased susceptibility to polymyxin B, we identified a number of M. avium mutants that are susceptible to the action of cathelicidin and are attenuated in both macrophages and mice. These findings are important because they unveil potential targets for therapy or prevention of the infection, as well as offering new insights into the pathogenicity of M. avium.

Abbreviations:

INH

isoniazid

MAC

Mycobacterium avium complex

OADC

oleic acid, albumin, glucose and catalase

PKS

polyketide synthase

1,25 vit D3

1,25-dihydroxyvitamin D3