Effects of lysosomal membrane protein depletion on the Salmonella-containing vacuole.

Salmonella is an intracellular bacterial pathogen that replicates within a membrane-bound vacuole in host cells. The major lysosomal membrane proteins 1 and 2 (LAMP-1 and LAMP-2) are recruited to the Salmonella-containing vacuole as well as Salmonella- associated filaments (Sifs) that emerge from the vacuole. LAMP-1 is a dominant membrane marker for the vacuole and Sifs. Its colocalization with both is dependent on a major secreted bacterial virulence protein, SifA. Here, we show that SifA is required for the recruitment of LAMP-2 and can be used as a second independent marker for both the bacterial vacuolar membrane and Sifs. Further, RNAi studies revealed that in LAMP-1 depleted cells, the bacteria remain membrane bound as measured by their association with LAMP-2 protein. In contrast, LAMP-2 depletion increased the amount of LAMP-1 free bacteria. Together, the data suggests that despite its abundance, LAMP-1 is not essential, but LAMP-2 may be partially important for the Salmonella-containing vacuolar membrane

LAMP-2 localizes to the SCV and Sifs.

<p>A. HeLa cells were infected with wild type bacteria expressing GFP. 18 hours post-infection, the monolayers were fixed and immuno-stained to detect LAMP-1 (i–iii) or LAMP-2 (iv–vi). Green bacteria co-localized with LAMP-1 and LAMP-2, suggesting that both are recruited to the SCV (indicated by the dotted line). Sifs, which are marked by arrows, are also labeled by LAMP-1 and LAMP-2. B. The extent of LAMP-2 labeling of the SCV is comparable to that seen for LAMP-1. Volocity image analysis software was used to quantitate the colocalization of GFP-<i>Salmonella</i> and LAMP-1 or LAMP-2. A two-tailed P value was obtained from the Volocity quantitation using the Student's T test. The P value is = 0.56 which indicates there is no significant difference between the colocalization of <i>Salmonella</i> with LAMP-1 or LAMP-2.</p

LAMP-2 recruitment is mediated by SifA.

<p>A. HeLa cells were infected with <i>ΔsseJ</i> (i–iii) or Δ<i>sseJ</i>/Δ<i>sifA</i> (iv–vi) mutants (green). 18 hours post-infection, the monolayers were fixed and imunno-stained for LAMP-2 (red). Δ<i>sseJ</i> bacteria colocalize with LAMP-2 (i–iii). <i>ΔsseJ/ΔsifA</i> mutants (green; iv–vi), show significantly reduced co-localization with LAMP-2. B. Volocity image analysis software was used to quantitate association of bacteria with LAMP-2 in <i>ΔsseJ/ΔsifA</i> and <i>ΔsseJ</i> mutants. A two-tailed P value was obtained from the Volocity quantitation using the Student's T test. The P value is 0.0001 which indicates there is significant difference between the colocalization of LAMP-2 with <i>ΔsseJ</i> versus <i>ΔsseJ/ΔsifA</i>.</p

LAMP-1 depleted <i>Salmonella</i> in LAMP-2 knockdown cells proliferate at an accelerated rate.

<p>Bar plot shows the number of <i>Salmonella</i> per infected cell. HeLa cells were transfected with RNAi oligos to knockdown LAMP-1 or LAMP-2. 48 hours after RNAi treatment, the cells were infected with GFP-<i>Salmonella</i> for 6 hours, then fixed and immuno-stained. The amount of bacteria per infected cell was counted and the results shown here. The cells that contained more than 20 bacteria were the ones in which the <i>Salmonella</i> were not contained in LAMP-1 vacuoles.</p

LAMP-2 knockdown increases prevalence of LAMP-1 depleted bacteria.

<p>A. RNAi treated HeLa cells were lysed 48 hours after RNAi treatment was initiated. Equal volumes of whole cell lysates were analyzed by Western for LAMP-1, LAMP-2 and actin (used as a loading control). B. LAMP-1:actin ratio relative to that in scrambled RNAi samples (set to 100) C. LAMP-2:actin ratio relative to that in scrambled RNAi samples (set to 100). The data in A–C show that LAMP-1 RNAi specifically knocks down LAMP-1 but not LAMP-2. In addition, LAMP-2 RNAi specifically knocks down LAMP-2 and not LAMP-1 D. HeLa cells were transfected with RNAi oligos to knockdown either LAMP-1 (i–iv) or LAMP-2 (v–ix). 48 hours after RNAi treatment, the cells were infected with <i>Salmonella</i> for 6 hours. The cells were then fixed and immuno-stained. LAMP-1 knockdown cells were immuno-stained for LAMP-2 and revealed consistent colocalization of <i>Salmonella</i> with LAMP-2 (iv). However, immuno-staining of LAMP-2 knockdown cells for LAMP-1 revealed reduced association of LAMP-1 in a subset of bacterial clusters (viii). Clusters of bacteria containing normal levels of LAMP-2 are shown by arrow heads. E. Volocity quantitation of bacteria association with LAMP-containing membrane in knockdown cells. A two-tailed P value was obtained from the Volocity quantitation using the Student's T test. Asterisk indicates the P value is less than 0.001 which indicates that the difference is significant.</p