Recombinant Salmonella paratyphi A Secreted effector protein pipB2 (pipB2)

What is PipB2 and how does it compare to its homologue PipB?

PipB2 is a type III secretion system (T3SS) effector protein secreted by Salmonella enterica into host cells. It differs significantly from its homologue PipB in its C-terminal region. While both proteins contain pentapeptide repeat regions, PipB2 possesses a unique C-terminal pentapeptide motif (LFNEF) that is absent in PipB. This structural difference appears to account for their divergent biological functions, particularly in the ability of PipB2 to affect late endosome/lysosome (LE/Lys) positioning .

What is the key structural feature of PipB2 that determines its functionality?

The C-terminal pentapeptide motif (LFNEF) is the critical structural feature of PipB2 that determines its functionality. Deletion of this C-terminal motif prevents peripheral targeting of PipB2 and eliminates its effect on organelle positioning. Experimental evidence shows that complementation studies using PipB2 constructs lacking this motif fail to restore the wild-type phenotype in Δ𝑝𝑖𝑝𝐵2 mutants .

How does PipB2 interact with host cell components?

PipB2 primarily interacts with the host cell's endocytic pathway, particularly targeting late endosomes and lysosomes. It reorganizes these compartments in mammalian cells, resulting in the centrifugal extension of lysosomal glycoprotein-rich membrane tubules (Salmonella-induced filaments or Sifs) along microtubules away from the Salmonella-containing vacuole (SCV). This interaction demonstrates PipB2's ability to override normal host cell vesicular trafficking processes .

What genetic approaches can be used to study PipB2 function?

Researchers can use multiple genetic approaches to study PipB2 function:

• Gene deletion: Creating Δ𝑝𝑖𝑝𝐵2 mutant strains to observe loss-of-function effects

• Complementation: Restoring the mutant phenotype using plasmid-expressed PipB2

• Domain deletion studies: Removing specific regions (like the LFNEF motif) to determine their importance

• Site-directed mutagenesis: Creating point mutations in key residues (such as T340A, L341A, F342A, N343A, E344A, and F345A) to assess their individual contributions

• Fusion constructs: Creating chimeric proteins (e.g., PipB-PipB2 fusions) to determine domain-specific functions
  The effectiveness of these approaches has been demonstrated in studies with S. Typhimurium, where complementation of Δ𝑝𝑖𝑝𝐵2 mutants with plasmid-borne PipB2 restored Sif extension to wild-type levels .

What microscopy techniques are most effective for visualizing PipB2 activity in infected cells?

Confocal microscopy is particularly effective for visualizing PipB2 activity in infected cells. The recommended protocol includes:

• Infecting cells with wild-type, mutant, or complemented Salmonella strains

• Fixing cells at appropriate time points (e.g., 12 hours post-infection for peak Sif formation)

• Immunostaining for LE/Lys markers (such as LAMP-1 or LAMP-2) to detect SCVs and Sifs

• Counterstaining for bacteria using anti-LPS antibodies and for microtubules using anti-β-tubulin antibodies

• Quantifying effects using the Sif extension index, which measures the area occupied by the Sif network as a percentage of the total cell area
  This approach provides both qualitative and quantitative assessment of PipB2 function in reorganizing host cell compartments.

How can researchers quantitatively assess PipB2's effect on Sif extension?

Researchers can quantitatively assess PipB2's effect on Sif extension using the Sif extension index method:

• Capture confocal microscopy images of infected cells stained for LAMP-2 (to visualize Sifs) and β-tubulin (to delineate total cell area)

• Measure the area occupied by the Sif network (LAMP-2 staining)

• Express this as a percentage of the total cell area (β-tubulin staining)

• Compare indices across different bacterial strains or conditions
  This method is more reliable than measuring individual Sif-tubule length as it accounts for the entire network and reduces bias. Using this approach, researchers have demonstrated that Sifs in Δ𝑝𝑖𝑝𝐵2-infected cells occupied significantly less area (40±14%) compared to wild-type (62±12%) or Δ𝑝𝑖𝑝𝐵 (63±13%) infected cells .

How does PipB2 contribute to Salmonella's intracellular lifestyle?

PipB2 contributes to Salmonella's intracellular lifestyle by remodeling the host cell's endosomal system to create an environment favorable for bacterial replication. Specifically:

• It mediates the extension of Salmonella-induced filaments (Sifs) along microtubules

• It influences the positioning of late endosomes/lysosomes, potentially redirecting resources to the SCV

• It helps establish and maintain the SCV as a replicative niche by modifying interactions with host endocytic compartments
  These functions are critical for Salmonella's ability to survive and replicate within host cells, a key aspect of its pathogenesis.

What are the differences in PipB2 function between Salmonella Typhimurium and Salmonella paratyphi A?

While the search results primarily focus on S. Typhimurium PipB2, the principles likely apply to S. paratyphi A with some key considerations:

• Sequence conservation: The C-terminal LFNEF motif responsible for PipB2 function in S. Typhimurium may be conserved in S. paratyphi A, suggesting similar mechanisms

• Host specificity: S. paratyphi A is human-restricted while S. Typhimurium has a broader host range, which might influence PipB2 adaptations

• Disease manifestation: S. paratyphi A causes paratyphoid fever while S. Typhimurium typically causes gastroenteritis in humans, suggesting potentially different roles for PipB2 in systemic versus localized infections
  Research specific to S. paratyphi A PipB2 would be needed to fully characterize these differences.

What is the relationship between PipB2 activity and Salmonella virulence?

PipB2 contributes to Salmonella virulence through:

• Facilitating SCV maturation by mediating interactions with host endocytic compartments

• Enabling efficient intracellular replication through remodeling of the host cell environment

• Potentially influencing nutrient acquisition by redirecting LE/Lys compartments

• Contributing to the establishment of a stable replicative niche protected from host defenses
  This relationship is evident in the significant impact of PipB2 on Sif extension, which correlates with Salmonella's ability to maintain its intracellular lifestyle .

How can researchers study PipB2's interactions with host cell proteins?

Researchers can study PipB2's interactions with host cell proteins using several advanced techniques:

• Co-immunoprecipitation: Using tagged versions of PipB2 (such as GFP-PipB2 or HA-PipB2) to pull down interacting host proteins

• Yeast two-hybrid screening: Identifying direct protein-protein interactions

• Proximity labeling methods: Using BioID or APEX2 fused to PipB2 to identify proteins in close proximity within living cells

• Co-expression studies: Expressing PipB2 with potential interactors like dominant-active Rab7 or Rab34 to assess functional relationships

• Mass spectrometry-based proteomics: Identifying changes in protein complex formation in the presence or absence of PipB2
  The search results indicate that co-expression studies have already revealed that PipB2 can overcome the effects of dominant-active Rab7 or Rab34 on LE/Lys positioning, suggesting it acts downstream or in a parallel pathway to these proteins .

What approaches can be used to study the trafficking and localization dynamics of PipB2 in real-time?

To study trafficking and localization dynamics of PipB2 in real-time, researchers can employ:

• Live-cell imaging with fluorescently tagged PipB2 (GFP-PipB2)

• Fluorescence recovery after photobleaching (FRAP) to measure protein mobility

• Single-particle tracking to follow individual PipB2-containing vesicles

• Correlative light and electron microscopy (CLEM) to precisely localize PipB2 at the ultrastructural level

• Optogenetic approaches to temporally control PipB2 activity and observe immediate effects
  These approaches would provide insight into the temporal aspects of PipB2 function, complementing the steady-state observations obtained through fixed-cell microscopy .

How do mutations in the LFNEF motif affect PipB2 function?

Mutations in the LFNEF motif significantly impair PipB2 function. The search results indicate that:

• Complete deletion of the C-terminal pentapeptide motif prevents peripheral targeting of PipB2

• This deletion also eliminates PipB2's effect on organelle positioning

• Individual alanine substitutions within this motif (T340A, L341A, F342A, N343A, E344A, and F345A) were created using site-directed mutagenesis to assess the contribution of each residue

• The PipB homologue that lacks this motif does not possess the same biological activity as PipB2
  These observations highlight the critical nature of the LFNEF motif for proper PipB2 function and localization .

What is the quantitative impact of PipB2 overexpression on host cell organelles?

The quantitative impact of PipB2 overexpression on host cell organelles has been assessed through multiple parameters:

• Overexpression of PipB2-2HA dramatically increases peripheral LAMP-1-positive vesicles (46±12% compared to 7.0±3.5% in wild type)

• This overexpression actually reduces Sif-positive cells (44±5.7% compared to 62±3.9% in wild type)

• Deletion constructs that remove the LFNEF motif restore these parameters to near wild-type levels
  These quantitative data demonstrate that PipB2 dosage is critical for its proper function in organelle positioning.

How does PipB2 interact with the host cell cytoskeleton?

PipB2 interacts with the host cell cytoskeleton, particularly microtubules, in several ways:

• It mediates the extension of Sifs along microtubules, as demonstrated by the reduced Sif extension in Δ𝑝𝑖𝑝𝐵2 mutants

• When overexpressed, it induces peripheral accumulation of LE/Lys compartments, suggesting it might recruit or activate motor proteins that drive transport along microtubules

• It potentially interacts with kinesin-1, a microtubule motor protein that drives transport toward the cell periphery

• Its activity results in centrifugal movement of vesicles, consistent with plus-end directed microtubule transport

• The reorganization of LE/Lys compartments by PipB2 is dependent on microtubules, as evidenced by the extension of Sifs along these cytoskeletal structures
  These interactions are critical for PipB2's role in reorganizing host cell compartments during Salmonella infection.

What techniques could be developed to enhance the study of S. paratyphi A PipB2 specifically?

To enhance the study of S. paratyphi A PipB2 specifically, researchers could develop:

• S. paratyphi A-specific genetic tools for generating clean deletion and complementation strains

• Human cell culture models that better represent the in vivo environment encountered by S. paratyphi A

• In vitro systems that reconstitute PipB2 interactions with human-specific host factors

• CRISPR-based approaches to modify host factors potentially interacting with PipB2

• Humanized mouse models for studying S. paratyphi A pathogenesis and PipB2 function in vivo

• Comparative genomics approaches to identify unique features of S. paratyphi A PipB2 compared to other serovars
  These techniques would address the current knowledge gap regarding S. paratyphi A-specific PipB2 functions.

What are the remaining unanswered questions about PipB2's mechanism of action?

Key unanswered questions about PipB2's mechanism of action include:

• What are the direct molecular binding partners of PipB2 in host cells?

• How does the LFNEF motif specifically mediate peripheral targeting?

• What is the structural basis for PipB2's interactions with host proteins?

• How is PipB2 activity regulated temporally during infection?

• What are the specific differences in PipB2 function between Salmonella serovars?

• How does PipB2 coordinate with other Salmonella effectors to modify the host cell environment?

• What is the evolutionary origin of the LFNEF motif and how is it conserved across Salmonella species? Addressing these questions would significantly advance our understanding of PipB2's role in Salmonella pathogenesis.