Pathogenic nontuberculous mycobacteria resist and inactivate cathelicidin: implication of a novel role for polar mycobacterial lipids

Includes bibliographic references.Nontuberculous mycobacteria (NTM) are a large group of environmental organisms with worldwide distribution, but only a relatively few are known to be pathogenic. Chronic, debilitating lung disease is the most common manifestation of NTM infection, which is often refractory to treatment. The incidence and prevalence of NTM lung disease are increasing in the United States and in many parts of the world. Hence, a more complete understanding of NTM pathogenesis will provide the foundation to develop innovative approaches to treat this recalcitrant disease. Herein, we domonstrate that several species of NTM show broad resistance to the antimicrobial peptide, cathelicidin (LL-37). Resistance to LL-37 was not significantly different between M. avium that contain serovar-specific glycopeptidolipid (GPL, M. aviumˢˢᴳᴾᴸ) and M.avium that do not (M. aviumᐞˢˢᴳᴾᴸ). Similarly, M. Abscessus containing non-specific GPL (M. abscessusⁿˢᴳᴾᴸ⁽⁺⁾) or lacking nsGPL (M. abscessusⁿˢᴳᴾᴸ⁽⁻⁾) remained equally resistant to LL-37. These findings would support the notion that GPL are not the components responsible for NTM resistance to LL-37. Unexpectedly, the growth of M. abscessusⁿˢᴳᴾᴸ⁽⁻⁾ increased with LL-37 or scrambled LL-37 peptide in a dose-dependent fashion. We also discovered that LL-37 exposed to NTM had reduced antimicrobial activity, and initial work indicates that this is likely due to inactivation of LL-37 by lipid component(s) of the NTM cell envelope. We conclude that pathogenic NTM resist and inactivate LL-37. The mechanism by which NTM circumvent the antimicrobial activity of LL-37 remains to be determined

ssGPL does not contribute to the resistance of <i>M</i>. <i>avium</i> to LL-37.

<p>CFU determination of <i>M</i>. <i>avium</i>^ssGPL and <i>M</i>. <i>avium</i>^ΔssGPL serovar 8 after incubation with 0, 10, 25, and 100 μg/ml LL-37 and 20 μg/ml gentamicin. ***p<0.0001. Data are the mean ± SEM of 3 independent experiments.</p

Loss of LL-37 activity after exposure to NTM or NTM-derived lipids.

<p><i>E</i>. <i>coli bioassays</i> were used to evaluate LL-37 activity. (A) LL-37 remaining in NTM (but not <i>Mtb</i>) culture supernatant no longer kills <i>E</i>. <i>coli</i>. (B) <i>E</i>. <i>coli</i> survives in untreated or boiled NTM culture supernatants to which fresh LL-37 was added. (C) <i>E</i>. <i>coli</i> survival following incubation with <i>M</i>. <i>abscessus</i> or <i>M</i>. <i>intracellulare</i> derived cell fractions. CM = cell membrane, CW = cell wall, ICW = insoluble cell wall fraction.</p

<i>E</i>. <i>coli</i> is susceptible to LL-37.

<p>(A) Log₁₀ CFU of <i>E</i>. <i>coli</i> after 4 hours of incubation with 0, 10, or 25 μg/ml of LL-37. **p< 0.001; ***p<0.0001. Data are the mean ± SEM of 6 independent experiments. (B) Images were taken of each serial dilution on LB agar from <i>E</i>. <i>coli</i> cultures incubated for 4 hours in the absence or presence of 25 μg/ml of LL-37.</p

LL-37 demonstrates broad-spectrum antimicrobial activity.

<p>Log₁₀ CFU after 1–8 hours of incubation of a (A) laboratory isolate of <i>Salmonella enteriditis</i> or clinical isolates of (B) <i>Salmonella enteriditis</i> (Uganda) or (C) <i>Salmonella non-typhi</i> (Nairobi) with 0–50 μg/ml of LL-37. *p< 0.01; **p< 0.001; ***p<0.0001. Data are the mean ± SEM of 3–6 independent experiments. (D) <i>Mtb</i> H37Rv were incubated with 10 μg/ml LL-37 and the percent change in CFU calculated after 96 hours incubation. n = 3 independent experiments.</p

nsGPL do not mediate the resistance of <i>M</i>. <i>abscessus</i> to LL-37.

<p>(A-B) CFU determination of <i>M</i>. <i>abscessus</i>^nsGPL(+) in the presence of the indicated concentrations of native LL-37 (p = 0.69). Data are the mean ± SEM of 4 independent experiments. (B) CFU determination of <i>M</i>. <i>abscessus</i>^nsGPL(-) in the presence of the indicated concentrations of native LL-37. Data are the mean ± SEM of 4 independent experiments. *p<0.01; **p< 0.001; ***p<0.0001. (C) Thin-layer chromatography demonstrates the presence and absence of GPL in <i>M</i>. <i>abscessus</i>^nsGPL(+) and <i>M</i>. <i>abscessus</i>^nsGPL(-), respectively. (D-E) CFU determination of <i>M</i>. <i>abscessus</i>^nsGPL(+) and <i>M</i>. <i>abscessus</i>^nsGPL(-), respectively in the presence of the indicated concentrations of scrambled LL-37 peptide. Data are the mean ± SEM of 3 independent experiments. *p<0.01; **p< 0.001; ***p<0.0001.</p

Chronic hyperoxia alters the early and late phases of the hypoxic ventilatory response in neonatal rats

Chronic hyperoxia during the first 1–4 postnatal weeks attenuates the hypoxic ventilatory response (HVR) subsequently measured in adult rats. Rather than focusing on this long-lasting plasticity, the present study considered the influence of hyperoxia on respiratory control during the neonatal period. Sprague-Dawley rats were born and raised in 60% O2 until studied at postnatal ages (P) of 4, 6–7, or 13–14 days. Ventilation and metabolism were measured in normoxia (21% O2) and acute hypoxia (12% O2) using head-body plethysmography and respirometry, respectively. Compared with age-matched rats raised in room air, the major findings were 1) diminished pulmonary ventilation and metabolic O2 consumption in normoxia at P4 and P6–7; 2) decreased breathing stability during normoxia; 3) attenuation of the early phase of the HVR at P6–7 and P13–14; and 4) a sustained increase in ventilation during hypoxia (vs. the normal biphasic HVR) at all ages studied. Attenuation of the early HVR likely reflects progressive impairment of peripheral arterial chemoreceptors while expression of a sustained HVR in neonates before P7 suggests that hyperoxia also induces plasticity within the central nervous system. Together, these results suggest a complex interaction between inhibitory and excitatory effects of hyperoxia on the developing respiratory control system

Inhibition of Nuclear Factor-Kappa B Activation Decreases Survival of <i>Mycobacterium tuberculosis</i> in Human Macrophages

<div><p></p><p>Nuclear factor-kappa B (NFκB) is a ubiquitous transcription factor that mediates pro-inflammatory responses required for host control of many microbial pathogens; on the other hand, NFκB has been implicated in the pathogenesis of other inflammatory and infectious diseases. Mice with genetic disruption of the p50 subunit of NFκB are more likely to succumb to <i>Mycobacterium tuberculosis</i> (<i>MTB</i>). However, the role of NFκB in host defense in humans is not fully understood. We sought to examine the role of NFκB activation in the immune response of human macrophages to <i>MTB</i>. Targeted pharmacologic inhibition of NFκB activation using BAY 11-7082 (BAY, an inhibitor of IκBα kinase) or an adenovirus construct with a dominant-negative IκBα significantly decreased the number of viable intracellular mycobacteria recovered from THP-1 macrophages four and eight days after infection. The results with BAY were confirmed in primary human monocyte-derived macrophages and alveolar macrophages. NFκB inhibition was associated with increased macrophage apoptosis and autophagy, which are well-established killing mechanisms of intracellular <i>MTB</i>. Inhibition of the executioner protease caspase-3 or of the autophagic pathway significantly abrogated the effects of BAY. We conclude that NFκB inhibition decreases viability of intracellular <i>MTB</i> in human macrophages via induction of apoptosis and autophagy.</p></div