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2015 ; 81
(2
): 726-35
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Autotransporter-based antigen display in bacterial ghosts
#MMPMID25398861
Hjelm A
; Söderström B
; Vikström D
; Jong WS
; Luirink J
; de Gier JW
Appl Environ Microbiol
2015[Jan]; 81
(2
): 726-35
PMID25398861
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Bacterial ghosts are empty cell envelopes of Gram-negative bacteria that can be
used as vehicles for antigen delivery. Ghosts are generated by releasing the
bacterial cytoplasmic contents through a channel in the cell envelope that is
created by the controlled production of the bacteriophage ?X174 lysis protein E.
While ghosts possess all the immunostimulatory surface properties of the original
host strain, they do not pose any of the infectious threats associated with live
vaccines. Recently, we have engineered the Escherichia coli autotransporter
hemoglobin protease (Hbp) into a platform for the efficient surface display of
heterologous proteins in Gram-negative bacteria, HbpD. Using the Mycobacterium
tuberculosis vaccine target ESAT6 (early secreted antigenic target of 6 kDa), we
have explored the application of HbpD to decorate E. coli and Salmonella ghosts
with antigens. The use of different promoter systems enabled the concerted
production of HbpD-ESAT6 and lysis protein E. Ghost formation was monitored by
determining lysis efficiency based on CFU, the localization of a set of cellular
markers, fluorescence microscopy, flow cytometry, and electron microscopy.
Hbp-mediated surface display of ESAT6 was monitored using a combination of a
protease accessibility assay, fluorescence microscopy, flow cytometry and
(immuno-)electron microscopy. Here, we show that the concerted production of HbpD
and lysis protein E in E. coli and Salmonella can be used to produce ghosts that
efficiently display antigens on their surface. This system holds promise for the
development of safe and cost-effective vaccines with optimal intrinsic adjuvant
activity and exposure of heterologous antigens to the immune system.
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