Identification of Mycobacterium tuberculosis Clinical Isolates with Altered Phagocytosis by Human Macrophages Due to a Truncated Lipoarabinomannan

Jordi B Torrelles,Rose Knaup,Avina Kolareth,Tatiana Slepushkina,Thomas M Kaufman,Peter Kang,Preston J Hill,Patrick J Brennan,Delphi Chatterjee,John T Belisle,James M Musser,Larry S Schlesinger

doi:10.1074/jbc.m806350200

Abstract

Phenotypically distinct clinical isolates of Mycobacterium tuberculosis are capable of altering the balance that exists between the pathogen and human host and ultimately the outcome of infection. This study has identified two M. tuberculosis strains (i.e. HN885 and HN1554) among a bank of clinical isolates with a striking defect in phagocytosis by primary human macrophages when compared with strain Erdman, a commonly used laboratory strain for studies of pathogenesis. Mass spectrometry in conjunction with NMR studies unequivocally confirmed that both HN885 and HN1554 contain truncated and more branched forms of mannose-capped lipoarabinomannan (ManLAM) with a marked reduction of their linear arabinan (corresponding mainly to the inner Araf-alpha(1-->5)-Araf unit) and mannan (with fewer 6-Manp residues and more substitutions in the linear Manp-alpha(1-->6)-Manp unit) domains. The truncation in the ManLAM molecules produced by strains HN885 and HN1554 led to a significant reduction in their surface availability. In addition, there was a marked reduction of higher order phosphatidyl-myo-inositol mannosides and the presence of dimycocerosates, triglycerides, and phenolic glycolipid in their cell envelope. Less exposed ManLAM and reduced higher order phosphatidyl-myo-inositol mannosides in strains HN885 and HN1554 resulted in their low association with the macrophage mannose receptor. Despite reduced phagocytosis, ingested bacilli replicated at a fast rate following serum opsonization. Our results provide evidence that the clinical spectrum of tuberculosis may be dictated not only by the host but also by the amounts and ratios of surface exposed mycobacterial adherence factors defined by strain genotype.

Highlights

Tuberculosis causes immense morbidity and mortality worldwide [1]
This study has identified two M. tuberculosis strains (i.e. HN885 and HN1554) among a bank of clinical isolates with a striking defect in phagocytosis by primary human macrophages when compared with strain Erdman, a commonly used laboratory strain for studies of pathogenesis
We randomly selected M. tuberculosis strains from the bank of PGG-1, -2, and -3 M. tuberculosis isolates described above to screen for alterations in bacterial association with primary human macrophages in order to better characterize microbial determinants important in this process

Summary

EXPERIMENTAL PROCEDURES

Chemical Reagents and Antibodies—All chemical reagents were of the highest grade from Sigma unless otherwise specified. Washed monolayers were incubated with either M. tuberculosis strain Erdman or HN885 in either RH containing 2.5% human serum or RHH at 37 °C for 2 h. Transmission Electron Microscopy (TEM) of Strains Erdman and HN885 in Macrophages—M. tuberculosis bacilli (1 ϫ 106) were added to MDM monolayers on glass coverslips and incubated for 2 h at 37 °C. Intracellular Growth of Strains Erdman and HN885 in Macrophages—For measurements of intracellular growth in macrophages, 12-day-old MDM monolayers were washed, and M. tuberculosis bacilli were added to MDMs (multiplicity of infection 1:1; duplicate wells) in either RH containing 2.5% serum or RHH and incubated for 2 h at 37 °C in 5% CO2 [27]. Infected monolayers were washed and either repleted with RH containing 1% human autologous serum and further incubated for 24, 48, or 72 h or lysed (0 h time point) as described [27]. Statistical Analysis—Statistical analyses were performed using GraphPad Prism version 4.0 (available on the World Wide Web)

RESULTS

Size and Molecular Composition

ErdLAM volumea

DISCUSSION

Strain Condition